U.S. patent application number 15/301491 was filed with the patent office on 2018-03-29 for method of target molecule characterisation using a molecular pore.
This patent application is currently assigned to Oxford Nanopore Technologies Ltd.. The applicant listed for this patent is Oxford Nanopore Technologies Ltd.. Invention is credited to James Anthony Clarke, Marion Louise Crawford, James White.
Application Number | 20180087101 15/301491 |
Document ID | / |
Family ID | 50776877 |
Filed Date | 2018-03-29 |
United States Patent
Application |
20180087101 |
Kind Code |
A9 |
Clarke; James Anthony ; et
al. |
March 29, 2018 |
METHOD OF TARGET MOLECULE CHARACTERISATION USING A MOLECULAR
PORE
Abstract
The invention relates to a new method of determining the
presence, absence or one or more characteristics of multiple
analytes. The invention concerns coupling a first analyte to a
membrane containing a detector and investigating the first analyte
using the detector. The invention also concerns coupling a second
analyte to the membrane and investigating the second analyte. The
first analyte is uncoupled from the membrane prior to investigating
the second analyte. The invention also relates to polynucleotide
sequencing.
Inventors: |
Clarke; James Anthony;
(Oxford, GB) ; Crawford; Marion Louise; (Oxford,
GB) ; White; James; (Oxford, GB) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Oxford Nanopore Technologies Ltd. |
Oxford |
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GB |
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Assignee: |
Oxford Nanopore Technologies
Ltd.
Oxford
GB
|
Prior
Publication: |
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Document Identifier |
Publication Date |
|
US 20170022557 A1 |
January 26, 2017 |
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Family ID: |
50776877 |
Appl. No.: |
15/301491 |
Filed: |
March 31, 2015 |
PCT Filed: |
March 31, 2015 |
PCT NO: |
PCT/GB2015/050992 PCKC 00 |
371 Date: |
October 3, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/GB2014/052737 |
Sep 10, 2014 |
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15301491 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6869 20130101;
C12Q 1/6869 20130101; C12Q 2565/631 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 4, 2014 |
GB |
1406155.0 |
Claims
1. A method for determining the presence, absence or one or more
characteristics of two or more analytes in two or more samples,
comprising: (a) coupling a first analyte in a first sample to a
membrane using one or more anchors; (b) allowing the first analyte
to interact with a detector present in the membrane and thereby
determining the presence, absence or one or more characteristics of
the first analyte; (c) uncoupling the first analyte from the
membrane; (d) coupling a second analyte in a second sample to the
membrane using one or more anchors; and (e) allowing the second
analyte to interact with a detector in the membrane and thereby
determining the presence, absence or one or more characteristics of
the second analyte.
2. A method according to claim 1, wherein (i) step (c) is performed
before step (d), (ii) step (d) is performed before step (c) or
(iii) steps (c) and (d) are performed at the same time.
3. A method according to claim 1, wherein the one or more anchors
comprise a polypeptide anchor and/or a hydrophobic anchor.
4. A method according to claim 3, wherein the hydrophobic anchor
comprises a lipid, fatty acid, sterol, carbon nanotube or amino
acid.
5. A method according to claim 1, wherein step (c) comprises
uncoupling the first analyte from the membrane by removing the one
or more anchors from the membrane.
6. A method according to claim 5, wherein step (c) comprises
contacting the one or more anchors with an agent which has a higher
affinity for the one or more anchors than the anchors have for the
membrane.
7. A method according to claim 6, wherein (i) the one or more
anchors comprises cholesterol and the agent is a cyclodextrin or a
derivative thereof or a lipid; (ii) the one or more anchors
comprises streptavidin, biotin or desthiobiotin and the agent is
biotin, desthiobiotin or streptavidin; or (iii) the one or more
anchors comprises a protein and the agent is an antibody or
fragment thereof which specifically binds to the protein.
8. A method according to claim 1, wherein step (c) comprises
contacting the one or more anchors with an agent which reduces
their ability to couple to the membrane.
9. A method according to claim 8, wherein (i) the one or more
anchors comprises cholesterol and the agent is cholesterol
dehydrogenase; (ii) the one or more anchors comprises a lipid and
the agent is a phospholipase; or (iii) the one or more anchors
comprises a protein and the agent is a proteinase or urea.
10. A method according to claim 1, wherein step (c) comprises
uncoupling the first analyte from the membrane by separating the
first analyte from the one or more anchors.
11. A method according to claim 1, wherein step (c) comprises
uncoupling the first analyte from the membrane by contacting the
first analyte and the one or more anchors with an agent which
competes with the first analyte for binding to the one or more
anchors.
12. A method according to claim 11, wherein the agent is a
polynucleotide which competes with the first analyte for
hybridisation to the one or more anchors.
13. A method according to claim 10, wherein step (c) comprises (i)
contacting the first analyte and the one or more anchors with urea,
tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT),
streptavidin or biotin, UV light, an enzyme or a binding agent;
(ii) heating the first analyte and one or more anchors; or (iii)
altering the pH.
14. (canceled)
15. (canceled)
16. A method according to claim 10, wherein step (d) comprises
coupling the second analyte to the membrane using the one or more
anchors that were separated from the first analyte.
17. A method according to claim 16, wherein steps (c) and (d)
comprise uncoupling the first analyte from the membrane by
contacting the membrane with the second analyte such that the
second analyte competes with the first analyte for binding to the
one or more anchors and replaces the first analyte.
18. (canceled)
19. A method according to claim 1, wherein between steps (c) and
(d) the method comprises removing at least some of the first sample
from the membrane.
20.-26. (canceled)
27. A method according to claim 1, wherein the detector comprises a
transmembrane pore.
28.-40. (canceled)
41. A method for uncoupling from a membrane an analyte coupled to
the membrane using cholesterol, comprising contacting the analyte
with a cyclodextrin or a derivative thereof and thereby uncoupling
the analyte from the membrane.
42.-44. (canceled)
45. A kit for determining the presence, absence or one or more
characteristics of two or more analytes in two or more samples
comprising (a) two or more anchors which are capable of coupling
the two or more analytes to a membrane and (b) one or more agents
which are capable of uncoupling at least one of the two or more
analytes from the membrane.
46.-48. (canceled)
49. A method according to claim 1, wherein the first and the second
analytes are polynucleotides and wherein the method is for
identifying or estimating the sequence of the first polynucleotide
and/or the second polynucleotide.
Description
FIELD OF THE INVENTION
[0001] The invention relates to a new method of determining the
presence, absence or one or more characteristics of multiple
analytes. The invention concerns coupling a first analyte to a
membrane containing a detector and investigating the first analyte
using the detector. The invention also concerns coupling a second
analyte to the membrane and investigating the second analyte. The
first analyte is uncoupled from the membrane prior to investigating
the second analyte. The invention also relates to polynucleotide
sequencing.
BACKGROUND OF THE INVENTION
[0002] There is currently a need for rapid and cheap polynucleotide
(e.g. DNA or RNA) sequencing and identification technologies across
a wide range of applications. Existing technologies are slow and
expensive mainly because they rely on amplification techniques to
produce large volumes of polynucleotide and require a high quantity
of specialist fluorescent chemicals for signal detection.
[0003] Transmembrane pores (nanopores) have great potential as
direct, electrical biosensors for polymers and a variety of small
molecules. In particular, recent focus has been given to nanopores
as a potential DNA sequencing technology.
[0004] When a potential is applied across a nanopore, there is a
change in the current flow when an analyte, such as a nucleotide,
resides transiently in the barrel for a certain period of time.
Nanopore detection of the nucleotide gives a current change of
known signature and duration. In the strand sequencing method, a
single polynucleotide strand is passed through the pore and the
identities of the nucleotides are derived. Strand sequencing can
involve the use of a polynucleotide binding protein to control the
movement of the polynucleotide through the pore.
[0005] It has previously been demonstrated that ultra low
concentration analyte delivery can be achieved by coupling the
analyte to a membrane in which the relevant detector is present.
This lowers by several orders of magnitude the amount of analyte
required in order to be detected (WO 2012/164270).
SUMMARY OF THE INVENTION
[0006] The inventors have surprisingly demonstrated that it is
possible to investigate multiple analytes in multiple samples by
successively coupling the analytes to a membrane in which a
detector is present. The first analyte is uncoupled from the
membrane prior to investigating the second analyte.
[0007] Accordingly, the invention provides a method for determining
the presence, absence or one or more characteristics of two or more
analytes in two or more samples, comprising: [0008] (a) coupling a
first analyte in a first sample to a membrane using one or more
anchors; [0009] (b) allowing the first analyte to interact with a
detector present in the membrane and thereby determining the
presence, absence or one or more characteristics of the first
analyte; [0010] (c) uncoupling the first analyte from the membrane;
[0011] (d) coupling a second analyte in a second sample to the
membrane using one or more anchors; and [0012] (e) allowing the
second analyte to interact with a detector in the membrane and
thereby determining the presence, absence or one or more
characteristics of the second analyte.
[0013] The invention also provides: [0014] a method for uncoupling
from a membrane an analyte coupled to the membrane using
cholesterol, comprising contacting the analyte with a cyclodextrin
or a derivative thereof and thereby uncoupling the analyte from the
membrane; and [0015] a kit for determining the presence, absence or
one or more characteristics of two or more analytes in two or more
samples comprising (a) a membrane, (b) two or more anchors which
are capable of coupling the two or more analytes to the membrane
and (c) one or more agents which are capable of uncoupling at least
one of the two or more analytes from the membrane.
DESCRIPTION OF THE FIGURES
[0016] FIG. 1 shows in section (1) the DNA template (SEQ ID NO: 31,
labelled A1 and SEQ ID NO: 47 labelled A2) used to prepare the DNA
used in Examples 2-4. Section (2) shows a cartoon representation of
construct X (described in full in Example 2 materials and
methods)--iSpC3 spacers are shown as crosses and four
5-nitroindoles as a grey box and the cholesterol tether as a grey
oval; label b=SEQ ID NO: 34, label c=SEQ ID NO: 35, label d=SEQ ID
NO: 39, label e=SEQ ID NO: 41. Section (3) shows a cartoon
representation of construct Y (described in full in Example 2
materials and methods)--iSpC3 spacers are shown as crosses and four
5-nitroindoles as a grey box and the cholesterol tether as a grey
oval; label b=SEQ ID NO: 34, label f=SEQ ID NO: 37, label g=SEQ ID
NO: 40, label h=SEQ ID NO: 30.
[0017] FIG. 2 shows the experimental time course (x-axis label=time
(s), y-axis label=percentage (%)) with the percentage of time the
nanopores are present in their unblocked state (shown as light
grey) compared to when a helicase DNA movement was occurring and
the nanopores were partially blocked by the DNA strand (shown as
black). DNA construct X was added at 2400 seconds as indicated by
the arrow labelled X. DNA construct Y was added at 7200 seconds as
indicated by the arrow labelled Y.
[0018] FIG. 3 shows part of the experimental time course (x-axis
label=time (s), y-axis label=percentage (%)) with the percentage of
time the nanopores are present in their unblocked state (shown as
light grey) compared to when a helicase DNA movement was occurring
and the nanopores were partially blocked by the DNA strand (shown
as black). DNA construct X was added at 2700 seconds as indicated
by the arrow labelled X. The buffer flush (10 mL) was at 7500
seconds as indicated by the arrow labelled F.
[0019] FIG. 4 shows part of the experimental time course (x-axis
label=time (s), y-axis label=percentage (%)) with the percentage of
time the nanopores are present in their unblocked state (shown as
light grey) compared to when a helicase DNA movement was occurring
and the nanopores were partially blocked by the DNA strand (shown
as black). DNA construct X was added at 2700 seconds as indicated
by the arrow labelled X. The 1 min methyl-.beta.-cyclodextrin
incubation and then flush (100 .mu.M, 150 .mu.L) was at 6900
seconds as indicated by the arrow labelled F.
[0020] FIG. 5 shows part of the experimental time course (x-axis
label=time (s), y-axis label=percentage (%)) with the percentage of
time the nanopores are present in their unblocked state (shown as
light grey) compared to when a helicase DNA movement was occurring
and the nanopores were partially blocked by the DNA strand (shown
as black). DNA construct X was added at 2400 seconds as indicated
by the arrow labelled X. The 10 min methyl-.beta.-cyclodextrin
incubation and then flush (100 .mu.M, 150 .mu.L) was between 6600
and 6900 seconds as indicated by the arrow labelled F and shown as
white boxes.
[0021] FIG. 6 shows part of the experimental time course (x-axis
label=time (s), y-axis label=percentage (%)) with the percentage of
time the nanopores are present in their unblocked state (shown as
light grey) compared to when a helicase DNA movement was occurring
and the nanopores were partially blocked by the DNA strand (shown
as black). DNA construct X was added at 2400 seconds as indicated
by the arrow labelled X. The 30 min methyl-.beta.-cyclodextrin
incubation and then flush (100 .mu.M, 150 .mu.L) was between 6300
and 8100 seconds as indicated by the arrow labelled F and shown as
white boxes.
[0022] FIG. 7 shows how the DNA construct used in Example 5 was
tethered to the membrane (labelled i). The strand of DNA which
translocated through the nanopore is labelled a (SEQ ID NO: 42
attached at its 3' end to four iSpC3 spacers (labelled as crosses)
which are attached at the opposite end to the 5' end of SEQ ID NO:
43). It was hybridised to two strands labelled b and c (SEQ ID NO:
44 and 45 respectively). SEQ ID NO: 45 was attached by its 3' end
to six iSp18 spacers (labelled d and shown as a dotted line) which
were attached at the opposite end to two thymines and a biotin
group (labelled f). The biotin group was bound to streptavidin
(labelled e) which also bound desthiobiotin (labelled g).
Desthiobiotin was attached to the 5' end of SEQ ID NO: 46 which had
a 3' cholesterol TEG (labelled h) at the opposite end.
[0023] FIG. 8 shows the current trace (y-axis label=Current (pA),
x-axis label=Time (s)) of the experiment described in Example 5.
The trace shows the coupling steps and the removal of the coupled
DNA using free biotin. *1 label corresponds to the addition of the
desthiobiotin extender, *2 corresponds to the addition of DNA
construct P, *3 corresponds to the addition of free biotin and *4
corresponds to the addition of the buffer flush.
[0024] FIG. 9 shows three zoomed in regions of the current trace
(all three traces have the following axes labels--y-axis
label=Current (pA), x-axis label=Time (s)) shown in FIG. 8. Traces
A, B and C are consecutive snap shots of part of the trace shown in
FIG. 8. *1 label corresponds to the addition of the desthiobiotin
extender, *2 corresponds to the addition of DNA construct P, *3
corresponds to the addition of free biotin and *4 corresponds to
the addition of the buffer flush.
DESCRIPTION OF THE SEQUENCE LISTING
[0025] SEQ ID NO: 1 shows the codon optimised polynucleotide
sequence encoding the MS-B1 mutant MspA monomer. This mutant lacks
the signal sequence and includes the following mutations: D90N,
D91N, D93N, D118R, D134R and E139K.
[0026] SEQ ID NO: 2 shows the amino acid sequence of the mature
form of the MS-B1 mutant of the MspA monomer. This mutant lacks the
signal sequence and includes the following mutations: D90N, D91N,
D93N, D118R, D134R and E139K.
[0027] SEQ ID NO: 3 shows the polynucleotide sequence encoding one
monomer of .alpha.-hemolysin-E111N/K147N (.alpha.-HL-NN; Stoddart
et al., PNAS, 2009; 106(19): 7702-7707).
[0028] SEQ ID NO: 4 shows the amino acid sequence of one monomer of
.alpha.-HL-NN.
[0029] SEQ ID NOs: 5 to 7 show the amino acid sequences of MspB, C
and D.
[0030] SEQ ID NO: 8 shows the polynucleotide sequence encoding the
Phi29 DNA polymerase.
[0031] SEQ ID NO: 9 shows the amino acid sequence of the Phi29 DNA
polymerase.
[0032] SEQ ID NO: 10 shows the codon optimised polynucleotide
sequence derived from the sbcB gene from E. coli. It encodes the
exonuclease I enzyme (EcoExo I) from E. coli.
[0033] SEQ ID NO: 11 shows the amino acid sequence of exonuclease I
enzyme (EcoExo I) from E. coli.
[0034] SEQ ID NO: 12 shows the codon optimised polynucleotide
sequence derived from the xthA gene from E. coli. It encodes the
exonuclease III enzyme from E. coli.
[0035] SEQ ID NO: 13 shows the amino acid sequence of the
exonuclease III enzyme from E. coli. This enzyme performs
distributive digestion of 5' monophosphate nucleosides from one
strand of double stranded DNA (dsDNA) in a 3'-5' direction. Enzyme
initiation on a strand requires a 5' overhang of approximately 4
nucleotides.
[0036] SEQ ID NO: 14 shows the codon optimised polynucleotide
sequence derived from the recJ gene from T. thermophilus. It
encodes the RecJ enzyme from T. thermophilus (TthRecJ-cd).
[0037] SEQ ID NO: 15 shows the amino acid sequence of the RecJ
enzyme from T. thermophilus (TthRecJ-cd). This enzyme performs
processive digestion of 5' monophosphate nucleosides from ssDNA in
a 5'-3' direction. Enzyme initiation on a strand requires at least
4 nucleotides.
[0038] SEQ ID NO: 16 shows the codon optimised polynucleotide
sequence derived from the bacteriophage lambda exo (redX) gene. It
encodes the bacteriophage lambda exonuclease.
[0039] SEQ ID NO: 17 shows the amino acid sequence of the
bacteriophage lambda exonuclease. The sequence is one of three
identical subunits that assemble into a trimer. The enzyme performs
highly processive digestion of nucleotides from one strand of
dsDNA, in a 5'-3' direction
(http://www.neb.com/nebecomm/products/productM0262.asp). Enzyme
initiation on a strand preferentially requires a 5' overhang of
approximately 4 nucleotides with a 5' phosphate.
[0040] SEQ ID NO: 18 shows the amino acid sequence of Hel308
Mbu.
[0041] SEQ ID NO: 19 shows the amino acid sequence of Hel308
Csy.
[0042] SEQ ID NO: 20 shows the amino acid sequence of Hel308
Tga.
[0043] SEQ ID NO: 21 shows the amino acid sequence of Hel308
Mhu.
[0044] SEQ ID NO: 22 shows the amino acid sequence of TraI Eco.
[0045] SEQ ID NO: 23 shows the amino acid sequence of XPD Mbu.
[0046] SEQ ID NO: 24 shows the amino acid sequence of Dda 1993.
[0047] SEQ ID NO: 25 shows the amino acid sequence of Trwc Cba.
[0048] SEQ ID NO: 26 shows a polynucleotide sequence used in
Example 1.
[0049] SEQ ID NO: 27 shows a polynucleotide sequence used in
Example 1. SEQ ID NO: 27 is attached at the 3' end to four iSp18
spacers which are attached at the opposite end to the 5' end of SEQ
ID NO: 28.
[0050] SEQ ID NO: 28 shows a polynucleotide sequence used in
Example 1. SEQ ID NO: 28 is attached at its 5' end to four iSp18
spacers which are attached at the opposite end to the 3' end of SEQ
ID NO: 27.
[0051] SEQ ID NO: 29 shows a polynucleotide sequence used in
Example 1.
[0052] SEQ ID NOs: 30 to 41 shows polynucleotide sequences used in
Example 2.
[0053] SEQ ID NO: 42 to 46 shows polynucleotide sequences used in
Example 5.
[0054] SEQ ID NO: 47 shows a polynucleotide sequence used in
Example 2.
DETAILED DESCRIPTION OF THE INVENTION
[0055] It is to be understood that different applications of the
disclosed products and methods may be tailored to the specific
needs in the art. It is also to be understood that the terminology
used herein is for the purpose of describing particular embodiments
of the invention only, and is not intended to be limiting.
[0056] In addition as used in this specification and the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the content clearly dictates otherwise. Thus, for
example, reference to "an analyte" includes two or more analytes,
reference to "a polynucleotide" includes two or more
polynucleotides, reference to "an anchor" refers to two or more
anchors, reference to "a helicase" includes two or more helicases,
reference to "a transmembrane pore" includes two or more pores and
the like.
[0057] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety.
Method of the Invention
[0058] The invention provides a method for determining the
presence, absence or one or more characteristics of two or more
analytes. The method comprises coupling a first analyte in a first
sample to a membrane using one or more anchors and allowing the
analyte to interact with a detector present in the membrane. The
presence, absence or one or more characteristics of the first
analyte is thereby determined. The method also comprises coupling a
second analyte in a second sample to the membrane using one or more
anchors and allowing the second analyte to interact with a detector
present in the membrane. The presence, absence or one or more
characteristics of the second analyte is thereby determined. The
first analyte may be uncoupled from the membrane before, after or
at the same time as the second analyte is coupled to the
membrane.
[0059] The inventors have surprisingly demonstrated that ultra low
concentration analyte delivery to a detector can be achieved by
coupling analytes to a membrane in which detector is present. This
lowers by several orders of magnitude the amount of analyte
required in order to be detected. The extent to which the amount of
analyte needed is reduced could not have been predicted.
[0060] In particular, the inventors surprisingly report an increase
in the capture of single stranded polynucleotide by .about.4 orders
of magnitude over that previously reported. As both the detector
and analyte are now on the same plane, then .about.10.sup.3 M
s.sup.-1 more interactions occur per second, as diffusion of both
molecules is in two dimensions rather than three dimensions. This
has dramatic implications on the sample preparation requirements
that are of key concern for diagnostic devices such as
next-generation sequencing systems.
[0061] In addition, coupling the analyte to a membrane has added
advantages for various nanopore-enzyme sequencing applications. In
strand sequencing, when the polynucleotide analyte is introduced
the pore may become blocked permanently or temporarily, preventing
the sequencing of the polynucleotide. When one end of the
polynucleotide analyte is localised away from the pore, for example
by coupling or tethering to the membrane, surprisingly it was found
that this temporary or permanent blocking is no longer observed. By
occupying one end of the polynucleotide by coupling it to the
membrane it also acts to effectively increase the analyte
concentration over the detector and so increase the sequencing
systems duty cycle.
[0062] The method is of course advantageous for detecting multiple
analytes that are present at low concentrations. The method
preferably allows the presence or one or more characteristics of
the two or more analytes to be determined when each analyte is
present at a concentration of from about 0.001 pM to about 1 nM,
such as less than 0.01 pM, less than 0.1 pM, less than 1 pM, less
than 10 pM or less than 100 pM.
[0063] The method of the invention is particularly advantageous for
polynucleotide sequencing because only small amounts of purified
polynucleotide can be obtained from human blood. The method
preferably allows estimating the sequence of, or allows sequencing
of, a polynucleotide that is present at a concentration of from
about 0.001 pM to about 1 nM, such as less than 0.01 pM, less than
0.1 pM, less than 1 pM, less than 10 pM or less than 100 pM. As
discussed in more detail below, the two or more analytes may be two
or more instances of the same analyte. This is advantageous in
polynucleotide sequencing because it allows the sequence of a
polynucleotide to be investigated more than once. This leads to
increased sequencing efficiency and accuracy.
[0064] Coupling one end of a polynucleotide to the membrane (even
temporarily) also means that the end will be prevented from
interfering with the nanopore-based sequencing process.
[0065] The method of the invention also has other advantages. The
method provides an alternative to the simultaneous measurement of
two or more analytes which removes the need to decouple the
measurement signals obtained from each analyte. The method enables
the sequential determination of two or more analytes wherein, for
example, the conditions required to determine each analyte differ,
thus making simultaneous measurement impractical. The method also
conveniently enables the measurement of two or more analytes using
the same membrane thus providing the possibility for multiple use
and extending the lifetime of the membrane.
Analytes
[0066] The method of the invention concerns determining the
presence, absence or one or more characteristics of two or more
analytes. Any number of analytes can be investigated. For instance,
the method of the invention may concern determining the presence,
absence or one or more characteristics of 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 50, 100 or more analytes. If three or more analytes are
investigated using the method of the invention, the second analyte
is also uncoupled from the membrane and the requisite number of
steps are added for the third analyte. The same is true for four or
more analytes.
[0067] The method of the invention may comprise determining or
measuring one or more characteristics of each analyte. The method
may involve determining or measuring two, three, four or five or
more characteristics of each analyte. The one or more
characteristics are preferably selected from (i) the size of the
analyte, (ii) the identity of the analyte, (iii) the secondary
structure of the analyte and (iv) whether or not the analyte is
modified. Any combination of (i) to (iv) may be measured in
accordance with the invention, such as {i}, {ii}, {iii}, {iv},
{i,ii}, {i,iii}, {i,iv}, {ii,iii}, {ii,iv}, {iii,iv}, {i,ii,iii},
{i,ii,iv}, {i,iii,iv}{ii,iii,iv} or {i,ii,iii,iv}. Different
combinations of (i) to (iv) may be measured for the first analyte
compared with the second analyte, including any of those
combinations listed above. The method preferably comprises
estimating the sequence of or sequencing a first polynucleotide
and/or a second polynucleotide.
[0068] Each analyte can be any substance. Suitable analytes
include, but are not limited to, metal ions, inorganic salts,
polymers, such as a polymeric acids or bases, dyes, bleaches,
pharmaceuticals, diagnostic agents, recreational drugs, explosives
and environmental pollutants.
[0069] The first analyte and/or second analyte can be an analyte
that is secreted from cells. Alternatively, the first analyte
and/or second analyte can be an analyte that is present inside
cells such that the analyte(s) must be extracted from the cells
before the invention can be carried out.
[0070] The first analyte and/or second analyte is preferably an
amino acid, peptide, polypeptide, a protein or a polynucleotide.
The amino acid, peptide, polypeptide or protein can be
naturally-occurring or non-naturally-occurring. The polypeptide or
protein can include within it synthetic or modified amino acids. A
number of different types of modification to amino acids are known
in the art. For the purposes of the invention, it is to be
understood that the first analyte and/or second analyte can be
modified by any method available in the art.
[0071] The protein can be an enzyme, antibody, hormone, growth
factor or growth regulatory protein, such as a cytokine. The
cytokine may be selected from an interleukin, preferably IFN-1,
IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 or IL-13, an interferon,
preferably IL-.gamma. or other cytokines such as TNF-.alpha.. The
protein may be a bacterial protein, fungal protein, virus protein
or parasite-derived protein. Before it is contacted with the
detector, the protein may be unfolded to form a polypeptide
chain.
[0072] The first analyte and/or second analyte is most preferably a
polynucleotide, such as a nucleic acid. Polynucleotides are
discussed in more detail below. A polynucleotide may be coupled to
the membrane at its 5' end or 3' end or at one or more intermediate
points along the strand. The polynucleotide can be single stranded
or double stranded as discussed below. The polynucleotide may be
circular. The polynucleotide may be an aptamer, a probe which
hybridises to microRNA or microRNA itself (Wang, Y. et al, Nature
Nanotechnology, 2011, 6, 668-674). The two polynucleotide analytes
may be polynucleotides which bind two proteins and may be used to
characterise the proteins, for instance to determine their
concentration.
[0073] When the analyte is a probe which hybridises to microRNA,
the probe may be coupled permanently or transiently to the
membrane. This is discussed in more detail below. The probe itself
may be adapted to couple directly to the membrane or may hybridise
to a complementary polynucleotide which has been adapted to couple
to the membrane. The analyte may be a complex of microRNA
hybridised to a probe where the probe has distinctive sequences or
barcodes enabling it to be identified unambiguously.
[0074] When the first analyte and/or second analyte is an aptamer,
the aptamer may be coupled permanently or transiently to the
membrane. The aptamer itself may be adapted to couple directly to
the membrane or may hybridise to a complementary polynucleotide
which has been adapted to couple to the membrane. The aptamer may
be bound or unbound to a protein analyte and the ultimate purpose
of detecting the aptamer may be to detect the presence, absence or
one or more characteristics of a protein analyte to which it
binds.
[0075] The first analyte and second analyte may be different from
one another. For instance, the first analyte may be a protein and
the second analyte may be a polynucleotide. Alternatively, the
first and second analytes may be different polynucleotides. In such
instances, there may be no need to remove at least part of the
first sample before adding the second sample. This is discussed in
more detail below. If the method concerns investigating three or
more analytes, they may all be different from one another or some
of them may be different from one another.
[0076] The first analyte and the second analyte may be two
instances of the same analyte. The first analyte may be identical
to the second analyte. This allows proofreading, particularly if
the analytes are polynucleotides. If the method concerns
investigating three or more analytes, they may all be three or more
instances of the same analyte or some of them may be separate
instances of the same analyte.
Polynucleotide
[0077] The first and/or second analyte is preferably a
polynucleotide. A polynucleotide, such as a nucleic acid, is a
macromolecule comprising two or more nucleotides. The
polynucleotide or nucleic acid may comprise any combination of any
nucleotides. The nucleotides can be naturally occurring or
artificial. One or more nucleotides in the polynucleotide can be
oxidized or methylated. One or more nucleotides in the
polynucleotide may be damaged. For instance, the polynucleotide may
comprise a pyrimidine dimer. Such dimers are typically associated
with damage by ultraviolet light and are the primary cause of skin
melanomas. One or more nucleotides in the polynucleotide may be
modified, for instance with a label or a tag. Suitable labels are
described below. The polynucleotide may comprise one or more
spacers.
[0078] A nucleotide typically contains a nucleobase, a sugar and at
least one phosphate group. The nucleobase and sugar form a
nucleoside.
[0079] The nucleobase is typically heterocyclic. Nucleobases
include, but are not limited to, purines and pyrimidines and more
specifically adenine (A), guanine (G), thymine (T), uracil (U) and
cytosine (C).
[0080] The sugar is typically a pentose sugar. Nucleotide sugars
include, but are not limited to, ribose and deoxyribose. The sugar
is preferably a deoxyribose.
[0081] The polynucleotide preferably comprises the following
nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and/or
thymidine (dT), deoxyguanosine (dG) and deoxycytidine (dC).
[0082] The nucleotide is typically a ribonucleotide or
deoxyribonucleotide. The nucleotide typically contains a
monophosphate, diphosphate or triphosphate. The nucleotide may
comprise more than three phosphates, such as 4 or 5 phosphates.
Phosphates may be attached on the 5' or 3' side of a nucleotide.
Nucleotides include, but are not limited to, adenosine
monophosphate (AMP), guanosine monophosphate (GMP), thymidine
monophosphate (TMP), uridine monophosphate (UMP), 5-methylcytidine
monophosphate, 5-hydroxymethylcytidine monophosphate, cytidine
monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic
guanosine monophosphate (cGMP), deoxyadenosine monophosphate
(dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine
monophosphate (dTMP), deoxyuridine monophosphate (dUMP),
deoxycytidine monophosphate (dCMP) and deoxymethylcytidine
monophosphate. The nucleotides are preferably selected from AMP,
TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP and dUMP.
[0083] A nucleotide may be abasic (i.e. lack a nucleobase). A
nucleotide may also lack a nucleobase and a sugar (i.e. is a C3
spacer).
[0084] The nucleotides in the polynucleotide may be attached to
each other in any manner. The nucleotides are typically attached by
their sugar and phosphate groups as in nucleic acids. The
nucleotides may be connected via their nucleobases as in pyrimidine
dimers.
[0085] The polynucleotide may be single stranded or double
stranded. At least a portion of the polynucleotide is preferably
double stranded.
[0086] The polynucleotide can be a nucleic acid, such as
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The
polynucleotide can comprise one strand of RNA hybridised to one
strand of DNA. The polynucleotide may be any synthetic nucleic acid
known in the art, such as peptide nucleic acid (PNA), glycerol
nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid
(LNA), bridged nucleic acid (BNA) or other synthetic polymers with
nucleotide side chains. The PNA backbone is composed of repeating
N-(2-aminoethyl)-glycine units linked by peptide bonds. The GNA
backbone is composed of repeating glycol units linked by
phosphodiester bonds. The TNA backbone is composed of repeating
threose sugars linked together by phosphodiester bonds. LNA is
formed from ribonucleotides as discussed above having an extra
bridge connecting the 2' oxygen and 4' carbon in the ribose
moiety.
[0087] The polynucleotide is most preferably ribonucleic nucleic
acid (RNA) or deoxyribonucleic acid (DNA).
[0088] The polynucleotide can be any length. For example, the
polynucleotide can be at least 10, at least 50, at least 100, at
least 150, at least 200, at least 250, at least 300, at least 400
or at least 500 nucleotides or nucleotide pairs in length. The
polynucleotide can be 1000 or more nucleotides or nucleotide pairs,
5000 or more nucleotides or nucleotide pairs in length or 100000 or
more nucleotides or nucleotide pairs in length.
Sample
[0089] Each analyte is typically present in any suitable sample.
The invention is typically carried out on two or more samples that
are known to contain or suspected to contain the analytes.
Alternatively, the invention may be carried out on two or more
samples to confirm the identity of two or more analytes whose
presence in the samples is known or expected.
[0090] The first sample and/or second sample may be a biological
sample. The invention may be carried out in vitro using at least
one sample obtained from or extracted from any organism or
microorganism. The organism or microorganism is typically archaeal,
prokaryotic or eukaryotic and typically belongs to one of the five
kingdoms: plantae, animalia, fungi, monera and protista. The
invention may be carried out in vitro on at least one sample
obtained from or extracted from any virus. The first sample and/or
second sample is preferably a fluid sample. The first sample and/or
second sample typically comprises a body fluid of the patient. The
first sample and/or second sample may be urine, lymph, saliva,
mucus or amniotic fluid but is preferably blood, plasma or serum.
Typically, the first sample and/or second sample is human in
origin, but alternatively it may be from another mammal animal such
as from commercially farmed animals such as horses, cattle, sheep,
fish, chickens or pigs or may alternatively be pets such as cats or
dogs. Alternatively, the first sample and/or second sample may be
of plant origin, such as a sample obtained from a commercial crop,
such as a cereal, legume, fruit or vegetable, for example wheat,
barley, oats, canola, maize, soya, rice, rhubarb, bananas, apples,
tomatoes, potatoes, grapes, tobacco, beans, lentils, sugar cane,
cocoa, cotton.
[0091] The first sample and/or second sample may be a
non-biological sample. The non-biological sample is preferably a
fluid sample. Examples of non-biological samples include surgical
fluids, water such as drinking water, sea water or river water, and
reagents for laboratory tests.
[0092] The first sample and/or second sample is typically processed
prior to being used in the invention, for example by centrifugation
or by passage through a membrane that filters out unwanted
molecules or cells, such as red blood cells. The first sample
and/or second sample may be measured immediately upon being taken.
The first sample and/or second sample may also be typically stored
prior to assay, preferably below -70.degree. C.
[0093] The first sample and second sample may be different from one
another. For instance, the first sample may be derived from a human
and the second sample may be derived from a virus. If the first and
second samples are different from one another, they may contain or
be suspected of containing the same first and second analytes. If
the method concerns investigating three or more samples, they may
all be different from one another or some of them may be different
from one another.
[0094] The first sample and the second sample are preferably two
instances of the same sample. The first sample is preferably
identical to the second sample. This allows proofreading,
particularly if the analytes are polynucleotides. If the method
concerns investigating three or more samples, they may all be three
or more instances of the same sample or some of them may be
separate instances of the same sample.
Membrane
[0095] Any membrane may be used in accordance with the invention.
Suitable membranes are well-known in the art. The membrane is
preferably an amphiphilic layer. An amphiphilic layer is a layer
formed from amphiphilic molecules, such as phospholipids, which
have both hydrophilic and lipophilic properties. The amphiphilic
molecules may be synthetic or naturally occurring. Non-naturally
occurring amphiphiles and amphiphiles which form a monolayer are
known in the art and include, for example, block copolymers
(Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). Block
copolymers are polymeric materials in which two or more monomer
sub-units are polymerized together to create a single polymer
chain. Block copolymers typically have properties that are
contributed by each monomer sub-unit. However, a block copolymer
may have unique properties that polymers formed from the individual
sub-units do not possess. Block copolymers can be engineered such
that one of the monomer sub-units is hydrophobic (i.e. lipophilic),
whilst the other sub-unit(s) are hydrophilic whilst in aqueous
media. In this case, the block copolymer may possess amphiphilic
properties and may form a structure that mimics a biological
membrane. The block copolymer may be a diblock (consisting of two
monomer sub-units), but may also be constructed from more than two
monomer sub-units to form more complex arrangements that behave as
amphiphiles. The copolymer may be a triblock, tetrablock or
pentablock copolymer. The membrane is preferably a triblock
copolymer membrane.
[0096] Archaebacterial bipolar tetraether lipids are naturally
occurring lipids that are constructed such that the lipid forms a
monolayer membrane. These lipids are generally found in
extremophiles that survive in harsh biological environments,
thermophiles, halophiles and acidophiles. Their stability is
believed to derive from the fused nature of the final bilayer. It
is straightforward to construct block copolymer materials that
mimic these biological entities by creating a triblock polymer that
has the general motif hydrophilic-hydrophobic-hydrophilic. This
material may form monomeric membranes that behave similarly to
lipid bilayers and encompasses a range of phase behaviours from
vesicles through to laminar membranes. Membranes formed from these
triblock copolymers hold several advantages over biological lipid
membranes. Because the triblock copolymer is synthesized, the exact
construction can be carefully controlled to provide the correct
chain lengths and properties required to form membranes and to
interact with pores and other proteins.
[0097] Block copolymers may also be constructed from sub-units that
are not classed as lipid sub-materials; for example a hydrophobic
polymer may be made from siloxane or other non-hydrocarbon based
monomers. The hydrophilic sub-section of block copolymer can also
possess low protein binding properties, which allows the creation
of a membrane that is highly resistant when exposed to raw
biological samples. This head group unit may also be derived from
non-classical lipid head-groups.
[0098] Triblock copolymer membranes also have increased mechanical
and environmental stability compared with biological lipid
membranes, for example a much higher operational temperature or pH
range. The synthetic nature of the block copolymers provides a
platform to customize polymer based membranes for a wide range of
applications.
[0099] In a preferred embodiment, the invention provides a method
for determining the presence, absence or one or more
characteristics of two or more analytes in two or more samples,
comprising (a) coupling a first analyte in a first sample to a
membrane using one or more anchors comprising a triblock copolymer,
optionally wherein the membrane is modified to facilitate the
coupling; (b) allowing the first analyte to interact with a
detector present in the membrane and thereby determining the
presence, absence or one or more characteristics of the first
analyte; (c) uncoupling the first analyte from the membrane; (d)
coupling a second analyte in a second sample to the membrane using
one or more anchors; and (e) allowing the second analyte to
interact with a detector in the membrane and thereby determining
the presence, absence or one or more characteristics of the second
analyte.
[0100] The membrane is most preferably one of the membranes
disclosed in International Application No. PCT/GB2013/052766 or
PCT/GB2013/052767.
[0101] The amphiphilic molecules may be chemically-modified or
functionalised to facilitate coupling of the analyte.
[0102] The amphiphilic layer may be a monolayer or a bilayer. The
amphiphilic layer is typically planar. The amphiphilic layer may be
curved. The amphiphilic layer may be supported.
[0103] Amphiphilic membranes are typically naturally mobile,
essentially acting as two dimensional fluids with lipid diffusion
rates of approximately 10.sup.-8 cm s-1. This means that the
detector and coupled analyte can typically move within an
amphiphilic membrane.
[0104] The membrane may be a lipid bilayer. Lipid bilayers are
models of cell membranes and serve as excellent platforms for a
range of experimental studies. For example, lipid bilayers can be
used for in vitro investigation of membrane proteins by
single-channel recording. Alternatively, lipid bilayers can be used
as biosensors to detect the presence of a range of substances. The
lipid bilayer may be any lipid bilayer. Suitable lipid bilayers
include, but are not limited to, a planar lipid bilayer, a
supported bilayer or a liposome. The lipid bilayer is preferably a
planar lipid bilayer. Suitable lipid bilayers are disclosed in
International Application No. PCT/GB08/000563 (published as WO
2008/102121), International Application No. PCT/GB08/004127
(published as WO 2009/077734) and International Application No.
PCT/GB2006/001057 (published as WO 2006/100484).
[0105] Methods for forming lipid bilayers are known in the art.
Suitable methods are disclosed in the Example. Lipid bilayers are
commonly formed by the method of Montal and Mueller (Proc. Natl.
Acad. Sci. USA., 1972; 69: 3561-3566), in which a lipid monolayer
is carried on aqueous solution/air interface past either side of an
aperture which is perpendicular to that interface. The lipid is
normally added to the surface of an aqueous electrolyte solution by
first dissolving it in an organic solvent and then allowing a drop
of the solvent to evaporate on the surface of the aqueous solution
on either side of the aperture. Once the organic solvent has
evaporated, the solution/air interfaces on either side of the
aperture are physically moved up and down past the aperture until a
bilayer is formed. Planar lipid bilayers may be formed across an
aperture in a membrane or across an opening into a recess.
[0106] The method of Montal & Mueller is popular because it is
a cost-effective and relatively straightforward method of forming
good quality lipid bilayers that are suitable for protein pore
insertion. Other common methods of bilayer formation include
tip-dipping, painting bilayers and patch-clamping of liposome
bilayers.
[0107] Tip-dipping bilayer formation entails touching the aperture
surface (for example, a pipette tip) onto the surface of a test
solution that is carrying a monolayer of lipid. Again, the lipid
monolayer is first generated at the solution/air interface by
allowing a drop of lipid dissolved in organic solvent to evaporate
at the solution surface. The bilayer is then formed by the
Langmuir-Schaefer process and requires mechanical automation to
move the aperture relative to the solution surface.
[0108] For painted bilayers, a drop of lipid dissolved in organic
solvent is applied directly to the aperture, which is submerged in
an aqueous test solution. The lipid solution is spread thinly over
the aperture using a paintbrush or an equivalent. Thinning of the
solvent results in formation of a lipid bilayer. However, complete
removal of the solvent from the bilayer is difficult and
consequently the bilayer formed by this method is less stable and
more prone to noise during electrochemical measurement.
[0109] Patch-clamping is commonly used in the study of biological
cell membranes. The cell membrane is clamped to the end of a
pipette by suction and a patch of the membrane becomes attached
over the aperture. The method has been adapted for producing lipid
bilayers by clamping liposomes which then burst to leave a lipid
bilayer sealing over the aperture of the pipette. The method
requires stable, giant and unilamellar liposomes and the
fabrication of small apertures in materials having a glass
surface.
[0110] Liposomes can be formed by sonication, extrusion or the
Mozafari method (Colas et al. (2007) Micron 38:841-847).
[0111] In a preferred embodiment, the lipid bilayer is formed as
described in International Application No. PCT/GB08/004127
(published as WO 2009/077734). Advantageously in this method, the
lipid bilayer is formed from dried lipids. In a most preferred
embodiment, the lipid bilayer is formed across an opening as
described in WO2009/077734 (PCT/GB08/004127).
[0112] A lipid bilayer is formed from two opposing layers of
lipids. The two layers of lipids are arranged such that their
hydrophobic tail groups face towards each other to form a
hydrophobic interior. The hydrophilic head groups of the lipids
face outwards towards the aqueous environment on each side of the
bilayer. The bilayer may be present in a number of lipid phases
including, but not limited to, the liquid disordered phase (fluid
lamellar), liquid ordered phase, solid ordered phase (lamellar gel
phase, interdigitated gel phase) and planar bilayer crystals
(lamellar sub-gel phase, lamellar crystalline phase).
[0113] Any lipid composition that forms a lipid bilayer may be
used. The lipid composition is chosen such that a lipid bilayer
having the required properties, such surface charge, ability to
support membrane proteins, packing density or mechanical
properties, is formed. The lipid composition can comprise one or
more different lipids. For instance, the lipid composition can
contain up to 100 lipids. The lipid composition preferably contains
1 to 10 lipids. The lipid composition may comprise
naturally-occurring lipids and/or artificial lipids.
[0114] The lipids typically comprise a head group, an interfacial
moiety and two hydrophobic tail groups which may be the same or
different. Suitable head groups include, but are not limited to,
neutral head groups, such as diacylglycerides (DG) and ceramides
(CM); zwitterionic head groups, such as phosphatidylcholine (PC),
phosphatidylethanolamine (PE) and sphingomyelin (SM); negatively
charged head groups, such as phosphatidylglycerol (PG);
phosphatidylserine (PS), phosphatidylinositol (PI), phosphatic acid
(PA) and cardiolipin (CA); and positively charged headgroups, such
as trimethylammonium-Propane (TAP). Suitable interfacial moieties
include, but are not limited to, naturally-occurring interfacial
moieties, such as glycerol-based or ceramide-based moieties.
Suitable hydrophobic tail groups include, but are not limited to,
saturated hydrocarbon chains, such as lauric acid (n-Dodecanolic
acid), myristic acid (n-Tetradecononic acid), palmitic acid
(n-Hexadecanoic acid), stearic acid (n-Octadecanoic) and arachidic
(n-Eicosanoic); unsaturated hydrocarbon chains, such as oleic acid
(cis-9-Octadecanoic); and branched hydrocarbon chains, such as
phytanoyl. The length of the chain and the position and number of
the double bonds in the unsaturated hydrocarbon chains can vary.
The length of the chains and the position and number of the
branches, such as methyl groups, in the branched hydrocarbon chains
can vary. The hydrophobic tail groups can be linked to the
interfacial moiety as an ether or an ester. The lipids may be
mycolic acid.
[0115] The lipids can also be chemically-modified. The head group
or the tail group of the lipids may be chemically-modified.
Suitable lipids whose head groups have been chemically-modified
include, but are not limited to, PEG-modified lipids, such as
1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene
glycol)-2000]; functionalised PEG Lipids, such as
1,2-Distearoyl-sn-Glycero-3
Phosphoethanolamine-N-[Biotinyl(Polyethylene Glycol)2000]; and
lipids modified for conjugation, such as
1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(succinyl) and
1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-(Biotinyl).
Suitable lipids whose tail groups have been chemically-modified
include, but are not limited to, polymerisable lipids, such as
1,2-bis(10,12-tricosadiynoyl)-sn-Glycero-3-Phosphocholine;
fluorinated lipids, such as
1-Palmitoyl-2-(16-Fluoropalmitoyl)-sn-Glycero-3-Phosphocholine;
deuterated lipids, such as
1,2-Dipalmitoyl-D62-sn-Glycero-3-Phosphocholine; and ether linked
lipids, such as 1,2-Di-O-phytanyl-sn-Glycero-3-Phosphocholine. The
lipids may be chemically-modified or functionalised to facilitate
coupling of the analyte.
[0116] The amphiphilic layer, for example the lipid composition,
typically comprises one or more additives that will affect the
properties of the layer. Suitable additives include, but are not
limited to, fatty acids, such as palmitic acid, myristic acid and
oleic acid; fatty alcohols, such as palmitic alcohol, myristic
alcohol and oleic alcohol; sterols, such as cholesterol,
ergosterol, lanosterol, sitosterol and stigmasterol;
lysophospholipids, such as
1-Acyl-2-Hydroxy-sn-Glycero-3-Phosphocholine; and ceramides.
[0117] In another preferred embodiment, the membrane is a solid
state layer. Solid state layers can be formed from both organic and
inorganic materials including, but not limited to, microelectronic
materials, insulating materials such as Si.sub.3N.sub.4,
Al.sub.2O.sub.3, and SiO, organic and inorganic polymers such as
polyamide, plastics such as Teflon.RTM. or elastomers such as
two-component addition-cure silicone rubber, and glasses. The solid
state layer may be formed from graphene. Suitable graphene layers
are disclosed in International Application No. PCT/US2008/010637
(published as WO 2009/035647).
[0118] The method is typically carried out using (i) an artificial
amphiphilic layer comprising a pore, (ii) an isolated,
naturally-occurring lipid bilayer comprising a pore, or (iii) a
cell having a pore inserted therein. The method is typically
carried out using an artificial amphiphilic layer, such as an
artificial triblock copolymer layer. The layer may comprise other
transmembrane and/or intramembrane proteins as well as other
molecules in addition to the pore. Suitable apparatus and
conditions are discussed below. The method of the invention is
typically carried out in vitro.
Coupling
[0119] Each analyte may be coupled to the membrane using any known
method. Each analyte is coupled to the membrane using one or more
anchors.
[0120] Coupling means that the analyte is intentionally linked with
the membrane using the one or more anchors. The method preferably
comprises specifically coupling the first analyte to the membrane
using the one or more anchors. The method preferably comprises
specifically coupling the second analyte to the membrane using the
one or more anchors. The first analyte and/or the second analyte is
preferably not coupled with the membrane via non-specific
interactions.
[0121] Each anchor comprises a group which couples (or binds) to
the adaptor and a group which couples (or binds) to the membrane.
Each anchor may covalently couple (or bind) to the adaptor and/or
the membrane.
[0122] Each analyte may be coupled to the membrane using any number
of anchors, such as 2, 3, 4 or more anchors. For instance, one
analyte may be coupled to the membrane using two anchors each of
which separately couples (or binds) to both the analyte and
membrane.
[0123] The one or more anchors may comprise one or more
polynucleotide binding proteins. Each anchor may comprise one or
more polynucleotide binding proteins. The polynucleotide binding
protein(s) may be any of those discussed below.
[0124] In some embodiments, the second analyte is coupled to the
membrane using the one or more anchors that were left behind in the
membrane following the uncoupling of the first analyte.
Alternatively, the second analyte is coupled to the membrane using
other (or separate) one or more anchors. The one or more anchors
used to couple the second analyte may be the same type of anchor
used to couple the first analyte or may be a different type of
anchor. This is discussed in more detail below.
[0125] If the membrane is an amphiphilic layer, such as a triblock
copolymer membrane, the one or more anchors preferably comprise a
polypeptide anchor present in the membrane and/or a hydrophobic
anchor present in the membrane. The hydrophobic anchor is
preferably a lipid, fatty acid, sterol, carbon nanotube,
polypeptide, protein or amino acid, for example cholesterol,
palmitate or tocopherol. In preferred embodiments, the one or more
anchors are not the detector.
[0126] The components of the membrane, such as the amphiphilic
molecules, copolymer or lipids, may be chemically-modified or
functionalised to form the one or more anchors. Examples of
suitable chemical modifications and suitable ways of
functionalising the components of the membrane are discussed in
more detail below. Any proportion of the membrane components may be
functionalized, for example at least 0.01%, at least 0.1%, at least
1%, at least 10%, at least 25%, at least 50% or 100%.
[0127] The first and/or second analyte may be coupled directly to
the membrane. The one or more anchors used to couple the first
analyte and/or the second analyte to the membrane preferably
comprise a linker. The one or more anchors may comprise one or
more, such as 2, 3, 4 or more, linkers. One linker may be used
couple more than one, such as 2, 3, 4 or more, analytes to the
membrane.
[0128] Preferred linkers include, but are not limited to, polymers,
such as polynucleotides, polyethylene glycols (PEGs),
polysaccharides and polypeptides. These linkers may be linear,
branched or circular. For instance, the linker may be a circular
polynucleotide. If the analyte is itself a polynucleotide, it may
hybridise to a complementary sequence on the circular
polynucleotide linker.
[0129] The one or more anchors or one or more linkers may comprise
a component that can be cut or broken down, such as a restriction
site or a photolabile group.
[0130] Functionalised linkers and the ways in which they can couple
molecules are known in the art. For instance, linkers
functionalised with maleimide groups will react with and attach to
cysteine residues in proteins. In the context of this invention,
the protein may be present in the membrane, may be the analyte
itself or may be used to couple (or bind) to the analyte. This is
discussed in more detail below.
[0131] Crosslinkage of analytes can be avoided using a "lock and
key" arrangement. Only one end of each linker may react together to
form a longer linker and the other ends of the linker each react
with the analyte or membrane respectively. Such linkers are
described in International Application No. PCT/GB10/000132
(published as WO 2010/086602).
[0132] The use of a linker is preferred in the sequencing
embodiments discussed below. If a polynucleotide analyte is
permanently coupled directly to the membrane in the sense that it
does not uncouple when interacting with the detector (i.e. does not
uncouple in step (b) or (e)), then some sequence data will be lost
as the sequencing run cannot continue to the end of the
polynucleotide due to the distance between the membrane and the
detector. If a linker is used, then the polynucleotide analyte can
be processed to completion.
[0133] The coupling may be permanent or stable. In other words, the
coupling may be such that the analyte remains coupled to the
membrane when interacting with the detector (i.e. does not uncouple
in step (b) or (e)).
[0134] The coupling may be transient. In other words, the coupling
may be such that the analyte may decouple from the membrane when
interacting with the detector (i.e. may uncouple in step (b) or
(e)). Typically, some instances of the first analyte remain coupled
to the membrane, for instance, because they do not interact with
the detector and so need to be uncoupled in step (c). For certain
applications, such as aptamer detection and polynucleotide
sequencing, the transient nature of the coupling is preferred. If a
permanent or stable linker is attached directly to either the 5' or
3' end of a polynucleotide and the linker is shorter than the
distance between the membrane and the transmembrane pore's channel,
then some sequence data will be lost as the sequencing run cannot
continue to the end of the polynucleotide. If the coupling is
transient, then when the coupled end randomly becomes free of the
membrane, then the polynucleotide can be processed to completion.
Chemical groups that form permanent/stable or transient links are
discussed in more detail below. The analyte may be transiently
coupled to an amphiphilic layer or triblock copolymer membrane
using cholesterol or a fatty acyl chain. Any fatty acyl chain
having a length of from 6 to 30 carbon atom, such as hexadecanoic
acid, may be used.
[0135] In preferred embodiments, a polynucleotide analyte, such as
a nucleic acid, is coupled to an amphiphilic layer such as a
triblock copolymer membrane or lipid bilayer. Coupling of nucleic
acids to synthetic lipid bilayers has been carried out previously
with various different tethering strategies. These are summarised
in Table 1 below.
TABLE-US-00001 TABLE 1 Anchor comprising Type of coupling Reference
Thiol Stable Yoshina-Ishii, C. and S. G. Boxer (2003). "Arrays of
mobile tethered vesicles on supported lipid bilayers." J Am Chem
Soc 125(13): 3696-7. Biotin Stable Nikolov, V., R. Lipowsky, et al.
(2007). "Behavior of giant vesicles with anchored DNA molecules."
Biophys J 92(12): 4356-68 Cholesterol Transient Pfeiffer, I. and F.
Hook (2004). "Bivalent cholesterol-based coupling of
oligonucletides to lipid membrane assemblies." J Am Chem Soc
126(33): 10224-5 Surfactant (e.g. Stable van Lengerich, B., R. J.
Rawle, et al. "Covalent Lipid, Palmitate, etc) attachment of lipid
vesicles to a fluid-supported bilayer allows observation of
DNA-mediated vesicle interactions." Langmuir 26(11): 8666-72
[0136] Synthetic polynucleotide analytes and/or linkers may be
functionalised using a modified phosphoramidite in the synthesis
reaction, which is easily compatible for the direct addition of
suitable anchoring groups, such as cholesterol, tocopherol,
palmitate, thiol, lipid and biotin groups. These different
attachment chemistries give a suite of options for attachment to
polynucleotides. Each different modification group couples the
polynucleotide in a slightly different way and coupling is not
always permanent so giving different dwell times for the analyte to
the membrane. The advantages of transient coupling are discussed
above.
[0137] Coupling of polynucleotides to a linker or to a
functionalised membrane can also be achieved by a number of other
means provided that a complementary reactive group or an anchoring
group can be added to the polynucleotide. The addition of reactive
groups to either end of a polynucleotide has been reported
previously. A thiol group can be added to the 5' of ssDNA or dsDNA
using T4 polynucleotide kinase and ATP.gamma.S (Grant, G. P. and P.
Z. Qin (2007). "A facile method for attaching nitroxide spin labels
at the 5' terminus of nucleic acids." Nucleic Acids Res 35(10):
e77). An azide group can be added to the 5'-phosphate of ssDNA or
dsDNA using T4 polynucleotide kinase and .gamma.-[2-Azidoethyl]-ATP
or .gamma.-[6-Azidohexyl]-ATP. Using thiol or Click chemistry a
tether, containing either a thiol, iodoacetamide OPSS or maleimide
group (reactive to thiols) or a DIBO (dibenzocyclooxtyne) or alkyne
group (reactive to azides), can be covalently attached to the
analyte. A more diverse selection of chemical groups, such as
biotin, thiols and fluorophores, can be added using terminal
transferase to incorporate modified oligonucleotides to the 3' of
ssDNA (Kumar, A., P. Tchen, et al. (1988). "Nonradioactive labeling
of synthetic oligonucleotide probes with terminal deoxynucleotidyl
transferase." Anal Biochem 169(2): 376-82). Streptavidin/biotin
and/or streptavidin/desthiobiotin coupling may be used for any
other analyte. The Examples below describes how a polynucleotide
can be coupled to a membrane using streptavidin/biotin and
streptavidin/desthiobiotin. It may also be possible that anchors
may be directly added to polynucleotides using terminal transferase
with suitably modified nucleotides (e.g. cholesterol or
palmitate).
[0138] The one or more anchors preferably couple the first analyte
and/or the second analyte to the membrane via hybridisation. The
hybridisation may be present in any part of the one or more
anchors, such as between the one or more anchors and the analyte,
within the one or more anchors or between the one or more anchors
and the membrane. Hybridisation in the one or more anchors allows
coupling in a transient manner as discussed above. For instance, a
linker may comprise two or more polynucleotides, such as 3, 4 or 5
polynucleotides, hybridised together. If the first analyte and/or
second analyte are themselves polynucleotides, the one or more
anchors may hybridise to the first polynucleotide analyte and/or
the second polynucleotide analyte. The one or more anchors may
hybridise directly to the polynucleotide analyte, directly to a Y
adaptor and/or leader sequence attached to the polynucleotide
analyte or directly to a hairpin loop adaptor attached to the
polynucleotide analyte (as discussed in more detail below).
Alternatively, the one or more anchors may be hybridised to one or
more, such as 2 or 3, intermediate polynucleotides (or "splints")
which are hybridised to the polynucleotide analyte, to a Y adaptor
and/or leader sequence attached to the polynucleotide analyte or to
a hairpin loop adaptor attached to the polynucleotide analyte (as
discussed in more detail below).
[0139] The one or more anchors may comprise a single stranded or
double stranded polynucleotide. One part of the anchor may be
ligated to a single stranded or double stranded polynucleotide
analyte. Ligation of short pieces of ssDNA have been reported using
T4 RNA ligase I (Troutt, A. B., M. G. McHeyzer-Williams, et al.
(1992). "Ligation-anchored PCR: a simple amplification technique
with single-sided specificity." Proc Natl Acad Sci USA 89(20):
9823-5). Alternatively, either a single stranded or double stranded
polynucleotide can be ligated to a double stranded polynucleotide
analyte and then the two strands separated by thermal or chemical
denaturation. To a double stranded polynucleotide, it is possible
to add either a piece of single stranded polynucleotide to one or
both of the ends of the duplex, or a double stranded polynucleotide
to one or both ends. For addition of single stranded
polynucleotides to the a double stranded polynucleotide, this can
be achieved using T4 RNA ligase I as for ligation to other regions
of single stranded polynucleotides. For addition of double stranded
polynucleotides to a double stranded polynucleotide analyte then
ligation can be "blunt-ended", with complementary 3' dA/dT tails on
the analyte and added polynucleotide respectively (as is routinely
done for many sample prep applications to prevent concatemer or
dimer formation) or using "sticky-ends" generated by restriction
digestion of the analyte and ligation of compatible adapters. Then,
when the duplex is melted, each single strand will have either a 5'
or 3' modification if a single stranded polynucleotide was used for
ligation or a modification at the 5' end, the 3' end or both if a
double stranded polynucleotide was used for ligation.
[0140] If the polynucleotide analyte is a synthetic strand, the one
or more anchors can be incorporated during the chemical synthesis
of the polynucleotide. For instance, the polynucleotide can be
synthesised using a primer having a reactive group attached to
it.
[0141] Adenylated polynucleotides are intermediates in ligation
reactions, where an adenosine-monophosphate is attached to the
5'-phosphate of the polynucleotide. Various kits are available for
generation of this intermediate, such as the 5' DNA Adenylation Kit
from NEB. By substituting ATP in the reaction for a modified
nucleotide triphosphate, then addition of reactive groups (such as
thiols, amines, biotin, azides, etc) to the 5' of a polynucleotide
can be possible. It may also be possible that anchors could be
directly added to polynucleotides using a 5' DNA adenylation kit
with suitably modified nucleotides (e.g. cholesterol or
palmitate).
[0142] A common technique for the amplification of sections of
genomic DNA is using polymerase chain reaction (PCR). Here, using
two synthetic oligonucleotide primers, a number of copies of the
same section of DNA can be generated, where for each copy the 5' of
each strand in the duplex will be a synthetic polynucleotide.
Single or multiple nucleotides can be added to 3' end of single or
double stranded DNA by employing a polymerase. Examples of
polymerases which could be used include, but are not limited to,
Terminal Transferase, Klenow and E. coli Poly(A) polymerase). By
substituting ATP in the reaction for a modified nucleotide
triphosphate then anchors, such as cholesterol, thiol, amine,
azide, biotin or lipid, can be incorporated into double stranded
polynucleotides. Therefore, each copy of the amplified
polynucleotide will contain an anchor.
[0143] Ideally, the analyte is coupled to the membrane without
having to functionalise the analyte. This can be achieved by
coupling the one or more anchors, such as a polynucleotide binding
protein or a chemical group, to the membrane and allowing the one
or more anchors to interact with the analyte or by functionalizing
the membrane. The one or more anchors may be coupled to the
membrane by any of the methods described herein. In particular, the
one or more anchors may comprise one or more linkers, such as
maleimide functionalised linkers.
[0144] In this embodiment, the analyte is typically RNA, DNA, PNA,
TNA or LNA and may be double or single stranded. This embodiment is
particularly suited to genomic DNA analytes.
[0145] The one or more anchors can comprise any group that couples
to, binds to or interacts with single or double stranded
polynucleotides, specific nucleotide sequences within the analyte
or patterns of modified nucleotides within the analyte, or any
other ligand that is present on the polynucleotide.
[0146] Suitable binding proteins for use in anchors include, but
are not limited to, E. coli single stranded binding protein, P5
single stranded binding protein, T4 gp32 single stranded binding
protein, the TOPO V dsDNA binding region, human histone proteins,
E. coli HU DNA binding protein and other archaeal, prokaryotic or
eukaryotic single stranded or double stranded polynucleotide (or
nucleic acid) binding proteins, including those listed below.
[0147] The specific nucleotide sequences could be sequences
recognised by transcription factors, ribosomes, endonucleases,
topoisomerases or replication initiation factors. The patterns of
modified nucleotides could be patterns of methylation or
damage.
[0148] The one or more anchors can comprise any group which couples
to, binds to, intercalates with or interacts with a polynucleotide
analyte. The group may intercalate or interact with the
polynucleotide analyte via electrostatic, hydrogen bonding or Van
der Waals interactions. Such groups include a lysine monomer,
poly-lysine (which will interact with ssDNA or dsDNA), ethidium
bromide (which will intercalate with dsDNA), universal bases or
universal nucleotides (which can hybridise with any polynucleotide
analyte) and osmium complexes (which can react to methylated
bases). A polynucleotide analyte may therefore be coupled to the
membrane using one or more universal nucleotides attached to the
membrane. Each universal nucleotide may be coupled to the membrane
using one or more linkers. The universal nucleotide preferably
comprises one of the following nucleobases: hypoxanthine,
4-nitroindole, 5-nitroindole, 6-nitroindole, formylindole,
3-nitropyrrole, nitroimidazole, 4-nitropyrazole,
4-nitrobenzimidazole, 5-nitroindazole, 4-aminobenzimidazole or
phenyl (C6-aromatic ring). The universal nucleotide more preferably
comprises one of the following nucleosides: 2'-deoxyinosine,
inosine, 7-deaza-2'-deoxyinosine, 7-deaza-inosine,
2-aza-deoxyinosine, 2-aza-inosine, 2-O'-methylinosine,
4-nitroindole 2'-deoxyribonucleoside, 4-nitroindole ribonucleoside,
5-nitroindole 2'-deoxyribonucleoside, 5-nitroindole ribonucleoside,
6-nitroindole 2'-deoxyribonucleoside, 6-nitroindole ribonucleoside,
3-nitropyrrole 2'-deoxyribonucleoside, 3-nitropyrrole
ribonucleoside, an acyclic sugar analogue of hypoxanthine,
nitroimidazole 2'-deoxyribonucleoside, nitroimidazole
ribonucleoside, 4-nitropyrazole 2'-deoxyribonucleoside,
4-nitropyrazole ribonucleoside, 4-nitrobenzimidazole
2'-deoxyribonucleoside, 4-nitrobenzimidazole ribonucleoside,
5-nitroindazole 2'-deoxyribonucleoside, 5-nitroindazole
ribonucleoside, 4-aminobenzimidazole 2'-deoxyribonucleoside,
4-aminobenzimidazole ribonucleoside, phenyl C-ribonucleoside,
phenyl C-2'-deoxyribosyl nucleoside, 2'-deoxynebularine,
2'-deoxyisoguanosine, K-2'-deoxyribose, P-2'-deoxyribose and
pyrrolidine. The universal nucleotide more preferably comprises
2'-deoxyinosine. The universal nucleotide is more preferably IMP or
dIMP. The universal nucleotide is most preferably dPMP
(2'-Deoxy-P-nucleoside monophosphate) or dKMP (N6-methoxy-2,
6-diaminopurine monophosphate).
[0149] The one or more anchors may couple to (or bind to) the
polynucleotide analyte via Hoogsteen hydrogen bonds (where two
nucleobases are held together by hydrogen bonds) or reversed
Hoogsteen hydrogen bonds (where one nucleobase is rotated through
180.degree. with respect to the other nucleobase). For instance,
the one or more anchors may comprise one or more nucleotides, one
or more oligonucleotides or one or more polynucleotides which form
Hoogsteen hydrogen bonds or reversed Hoogsteen hydrogen bonds with
the polynucleotide analyte. These types of hydrogen bonds allow a
third polynucleotide strand to wind around a double stranded helix
and form a triplex. The one or more anchors may couple to (or bind
to) a double stranded polynucleotide analyte by forming a triplex
with the double stranded duplex.
[0150] In this embodiment at least 1%, at least 10%, at least 25%,
at least 50% or 100% of the membrane components may be
functionalized.
[0151] Where the one or more anchors comprise a protein, they may
be able to anchor directly into the membrane without further
functonalisation, for example if it already has an external
hydrophobic region which is compatible with the membrane. Examples
of such proteins include, but are not limited to, transmembrane
proteins, intramembrane proteins and membrane proteins.
Alternatively the protein may be expressed with a genetically fused
hydrophobic region which is compatible with the membrane. Such
hydrophobic protein regions are known in the art.
[0152] The one or more anchors are preferably mixed with the
analyte before contacting with the membrane, but the one or more
anchors may be contacted with the membrane and subsequently
contacted with the analyte.
[0153] In another aspect the analyte may be functionalised, using
methods described above, so that it can be recognised by a specific
binding group. Specifically the analyte may be functionalised with
a ligand such as biotin (for binding to streptavidin), amylose (for
binding to maltose binding protein or a fusion protein), Ni-NTA
(for binding to poly-histidine or poly-histidine tagged proteins)
or peptides (such as an antigen).
[0154] According to a preferred embodiment, the one or more anchors
may be used to couple a polynucleotide analyte to the membrane when
the analyte is attached to a leader sequence which preferentially
threads into the pore. Leader sequences are discussed in more
detail below. Preferably, the polynucleotide analyte is attached
(such as ligated) to a leader sequence which preferentially threads
into the pore. Such a leader sequence may comprise a homopolymeric
polynucleotide or an abasic region. The leader sequence is
typically designed to hybridise to the one or more anchors either
directly or via one or more intermediate polynucleotides (or
splints). In such instances, the one or more anchors typically
comprise a polynucleotide sequence which is complementary to a
sequence in the leader sequence or a sequence in the one or more
intermediate polynucleotides (or splints). In such instances, the
one or more splints typically comprise a polynucleotide sequence
which is complementary to a sequence in the leader sequence.
[0155] Any of the methods discussed above for coupling
polynucleotides to membranes, such as amphiphilic layers, can of
course be applied to other analyte and membrane combinations. In
some embodiments, an amino acid, peptide, polypeptide or protein is
coupled to an amphiphilic layer, such as a triblock copolymer layer
or lipid bilayer. Various methodologies for the chemical attachment
of such analytes are available. An example of a molecule used in
chemical attachment is EDC
(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride).
Reactive groups can also be added to the 5' of polynucleotides
using commercially available kits (Thermo Pierce, Part No. 22980).
Suitable methods include, but are not limited to, transient
affinity attachment using histidine residues and Ni-NTA, as well as
more robust covalent attachment by reactive cysteines, lysines or
non natural amino acids.
Detector
[0156] Steps (b) and (e) comprise allowing the first analyte and
second analyte respectively to interact with a detector present in
the membrane and thereby determining the presence, absence or one
or more characteristics of the first analyte and second analyte
respectively. The detector in each step may be different. The
detector in each step is typically the same. For instance, both the
first and second analytes are preferably allowed to interact with a
transmembrane pore, preferably the same transmembrane pore.
[0157] The coupling of the first analyte and/or the second analyte
is not essential for the analyte to interact with the detector. The
coupling allows ultra low concentration analyte delivery to the
detector.
[0158] The detector can be any structure that provides a readable
signal in response to the presence, the absence or the one or more
characteristics of the first and/or second analyte. The detector
can be any structure that provides a readable signal in response to
the presence or the absence of the first and/or second analyte.
Suitable detectors are known in the art. They include, but are not
limited to transmembrane pores, tunnelling electrodes, classis
electrodes, nanotubes, FETs (field-effect transistors) and optical
detectors, such as atomic force microscopes (AFMs) and scanning
tunneling microscopes (STMs).
[0159] A variety of different types of measurements may be made.
This includes without limitation: electrical measurements and
optical measurements. Possible electrical measurements include:
current measurements, impedance measurements, tunnelling
measurements (Ivanov A P et al., Nano Lett. 2011 Jan. 12;
11(1):279-85), and FET measurements (International Application WO
2005/124888). Optical measurements may be combined with electrical
measurements (Soni G V et al., Rev Sci Instrum. 2010 January;
81(1):014301). The measurement may be a transmembrane current
measurement such as measurement of ionic current flowing through
the pore.
[0160] Electrical measurements may be made using standard single
channel recording equipment as describe in Stoddart D et al., Proc
Natl Acad Sci, 12; 106(19):7702-7, Lieberman K R et al, J Am Chem
Soc. 2010; 132(50):17961-72, and International Application WO
2000/28312. Alternatively, electrical measurements may be made
using a multi-channel system, for example as described in
International Application WO 2009/077734 and International
Application WO 2011/067559.
[0161] The method is preferably carried out with a potential
applied across the membrane. The applied potential may be a voltage
potential. Alternatively, the applied potential may be a chemical
potential. An example of this is using a salt gradient across a
membrane, such as an amphiphilic layer. A salt gradient is
disclosed in Holden et al., J Am Chem Soc. 2007 Jul. 11;
129(27):8650-5. In some instances, the current passing through the
detector (or pore) as a polynucleotide analyte moves with respect
to the pore is used to estimate or determine the sequence of the
polynucleotide. This is strand sequencing.
[0162] In other preferred embodiments, the detector does not detect
the analyte using fluorescent means.
[0163] The detector preferably comprises a transmembrane pore. A
transmembrane pore is a structure that crosses the membrane to some
degree. It permits hydrated ions driven by an applied potential to
flow across or within the membrane. The transmembrane pore
typically crosses the entire membrane so that hydrated ions may
flow from one side of the membrane to the other side of the
membrane. However, the transmembrane pore does not have to cross
the membrane. It may be closed at one end. For instance, the pore
may be a well, gap, channel, trench or slit in the membrane along
which or into which hydrated ions may flow.
[0164] If the detector is a pore, step (b) preferably comprises (i)
allowing the first analyte to interact with the detector and (ii)
measuring the current passing through the detector during the
interaction and thereby determining the presence, absence or one or
more characteristics of the first analyte and/or step (e) comprises
(i) allowing the second analyte to interact with the detector and
(ii) measuring the current passing through the detector during the
interaction and thereby determining the presence, absence or one or
more characteristics of the second analyte.
[0165] The first or second analyte is present if the current flows
through the pore in a manner specific for the analyte (i.e. if a
distinctive current associated with the analyte is detected flowing
through the pore). The first or second analyte is absent if the
current does not flow through the pore in a manner specific for the
analyte. Similarly, the characteristics of the analyte can be
determined using the current flowing through the pore during the
interaction.
[0166] The invention therefore involves nanopore sensing of an
analyte. The invention can be used to differentiate analytes of
similar structure on the basis of the different effects they have
on the current passing through the pore. The invention can also be
used to measure the concentration of a particular analyte in a
sample.
[0167] The invention may also be used in a sensor that uses many or
thousands of pores in bulk sensing applications.
[0168] During the interaction between the first or second analyte
and the pore, the analyte affects the current flowing through the
pore in a manner specific for that analyte. For example, a
particular analyte will reduce the current flowing through the pore
for a particular mean time period and to a particular extent. In
other words, the current flowing through the pore is distinctive
for a particular analyte. Control experiments may be carried out to
determine the effect a particular analyte has on the current
flowing through the pore. Results from carrying out the method of
the invention on a test sample can then be compared with those
derived from such a control experiment in order to identify a
particular analyte in the sample, determine whether a particular
analyte is present in the sample or determine the characteristics
of each analyte. The frequency at which the current flowing through
the pore is affected in a manner indicative of a particular analyte
can be used to determine the concentration of that analyte in the
sample.
[0169] Any transmembrane pore may be used in the invention. The
pore may be biological or artificial. Suitable pores include, but
are not limited to, protein pores, polynucleotide pores and solid
state pores. The pore may be a DNA origami pore (Langecker et al.,
Science, 2012; 338: 932-936).
[0170] The transmembrane pore is preferably a transmembrane protein
pore. A transmembrane protein pore is a polypeptide or a collection
of polypeptides that permits hydrated ions, such as analyte, to
flow from one side of a membrane to the other side of the membrane.
In the present invention, the transmembrane protein pore is capable
of forming a pore that permits hydrated ions driven by an applied
potential to flow from one side of the membrane to the other. The
transmembrane protein pore preferably permits analyte such as
nucleotides to flow from one side of the membrane, such as a
triblock copolymer membrane, to the other. The transmembrane
protein pore allows a polynucleotide, such as DNA or RNA, to be
moved through the pore.
[0171] The transmembrane protein pore may be a monomer or an
oligomer. The pore is preferably made up of several repeating
subunits, such as at least 3, at least 4, at least 5, at least 6,
at least 7, at least 8, at least 9, at least 10, at least 11, at
least 12, at least 13 or at least 14 subunits, such as 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13 or 14 subunits. The pore is preferably a
hexameric, heptameric, octameric or nonameric pore. The pore may be
a homo-oligomer or a hetero-oligomer.
[0172] The transmembrane protein pore typically comprises a barrel
or channel through which the ions may flow. The subunits of the
pore typically surround a central axis and contribute strands to a
transmembrane .beta. barrel or channel or a transmembrane
.alpha.-helix bundle or channel.
[0173] The barrel or channel of the transmembrane protein pore
typically comprises amino acids that facilitate interaction with
analyte, such as nucleotides, polynucleotides or nucleic acids.
These amino acids are preferably located near a constriction of the
barrel or channel. The transmembrane protein pore typically
comprises one or more positively charged amino acids, such as
arginine, lysine or histidine, or aromatic amino acids, such as
tyrosine or tryptophan. These amino acids typically facilitate the
interaction between the pore and nucleotides, polynucleotides or
nucleic acids.
[0174] Transmembrane protein pores for use in accordance with the
invention can be derived from .beta.-barrel pores or .alpha.-helix
bundle pores. .beta.-barrel pores comprise a barrel or channel that
is formed from .beta.-strands. Suitable .beta.-barrel pores
include, but are not limited to, .beta.-toxins, such as
.alpha.-hemolysin, anthrax toxin and leukocidins, and outer
membrane proteins/porins of bacteria, such as Mycobacterium
smegmatis porin (Msp), for example MspA, MspB, MspC or MspD,
lysenin, outer membrane porin F (OmpF), outer membrane porin G
(OmpG), outer membrane phospholipase A and Neisseria
autotransporter lipoprotein (NalP). .alpha.-helix bundle pores
comprise a barrel or channel that is formed from .alpha.-helices.
Suitable .alpha.-helix bundle pores include, but are not limited
to, inner membrane proteins and .alpha., outer membrane proteins,
such as WZA and ClyA toxin. The transmembrane pore may be derived
from lysenin. Suitable pores derived from lysenin are disclosed in
International Application No. PCT/GB2013/050667 (published as WO
2013/153359). The transmembrane pore may be derived from Msp or
from .alpha.-hemolysin (.alpha.-HL).
[0175] The transmembrane protein pore is preferably derived from
Msp, preferably from MspA. Such a pore will be oligomeric and
typically comprises 7, 8, 9 or 10 monomers derived from Msp. The
pore may be a homo-oligomeric pore derived from Msp comprising
identical monomers. Alternatively, the pore may be a
hetero-oligomeric pore derived from Msp comprising at least one
monomer that differs from the others. Preferably the pore is
derived from MspA or a homolog or paralog thereof.
[0176] A monomer derived from Msp typically comprises the sequence
shown in SEQ ID NO: 2 or a variant thereof. SEQ ID NO: 2 is the
MS-(B1)8 mutant of the MspA monomer. It includes the following
mutations: D90N, D91N, D93N, D118R, D134R and E139K. A variant of
SEQ ID NO: 2 is a polypeptide that has an amino acid sequence which
varies from that of SEQ ID NO: 2 and which retains its ability to
form a pore. The ability of a variant to form a pore can be assayed
using any method known in the art. For instance, the variant may be
inserted into an amphiphilic layer along with other appropriate
subunits and its ability to oligomerise to form a pore may be
determined. Methods are known in the art for inserting subunits
into membranes, such as amphiphilic layers. For example, subunits
may be suspended in a purified form in a solution containing a
triblock copolymer membrane such that it diffuses to the membrane
and is inserted by binding to the membrane and assembling into a
functional state. Alternatively, subunits may be directly inserted
into the membrane using the "pick and place" method described in M.
A. Holden, H. Bayley. J. Am. Chem. Soc. 2005, 127, 6502-6503 and
International Application No. PCT/GB2006/001057 (published as WO
2006/100484).
[0177] Over the entire length of the amino acid sequence of SEQ ID
NO: 2, a variant will preferably be at least 50% homologous to that
sequence based on amino acid identity. More preferably, the variant
may be at least 55%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90% and more
preferably at least 95%, 97% or 99% homologous based on amino acid
identity to the amino acid sequence of SEQ ID NO: 2 over the entire
sequence. There may be at least 80%, for example at least 85%, 90%
or 95%, amino acid identity over a stretch of 100 or more, for
example 125, 150, 175 or 200 or more, contiguous amino acids ("hard
homology").
[0178] Standard methods in the art may be used to determine
homology. For example the UWGCG Package provides the BESTFIT
program which can be used to calculate homology, for example used
on its default settings (Devereux et al (1984) Nucleic Acids
Research 12, p 387-395). The PILEUP and BLAST algorithms can be
used to calculate homology or line up sequences (such as
identifying equivalent residues or corresponding sequences
(typically on their default settings)), for example as described in
Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. F et al
(1990) J Mol Biol 215:403-10. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
[0179] SEQ ID NO: 2 is the MS-(B1)8 mutant of the MspA monomer. The
variant may comprise any of the mutations in the MspB, C or D
monomers compared with MspA. The mature forms of MspB, C and D are
shown in SEQ ID NOs: 5 to 7. In particular, the variant may
comprise the following substitution present in MspB: A138P. The
variant may comprise one or more of the following substitutions
present in MspC: A96G, N102E and A138P. The variant may comprise
one or more of the following mutations present in MspD: Deletion of
G1, L2V, E5Q, L8V, D13G, W21A, D22E, K47T, I49H, I68V, D91G, A96Q,
N102D, S103T, V104I, S136K and G141A. The variant may comprise
combinations of one or more of the mutations and substitutions from
Msp B, C and D. The variant preferably comprises the mutation L88N.
A variant of SEQ ID NO: 2 has the mutation L88N in addition to all
the mutations of MS-B1 and is called MS-(B2)8. The pore used in the
invention is preferably MS-(B2)8. A variant of SEQ ID NO: 2 has the
mutations G75S/G77S/L88N/Q126R in addition to all the mutations of
MS-B1 and is called MS-B2C. The pore used in the invention is
preferably MS-(B2)8 or MS-(B2C)8.
[0180] Amino acid substitutions may be made to the amino acid
sequence of SEQ ID NO: 2 in addition to those discussed above, for
example up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions.
Conservative substitutions replace amino acids with other amino
acids of similar chemical structure, similar chemical properties or
similar side-chain volume. The amino acids introduced may have
similar polarity, hydrophilicity, hydrophobicity, basicity,
acidity, neutrality or charge to the amino acids they replace.
Alternatively, the conservative substitution may introduce another
amino acid that is aromatic or aliphatic in the place of a
pre-existing aromatic or aliphatic amino acid.
[0181] One or more amino acid residues of the amino acid sequence
of SEQ ID NO: 2 may additionally be deleted from the polypeptides
described above. Up to 1, 2, 3, 4, 5, 10, 20 or 30 residues may be
deleted, or more.
[0182] Variants may include fragments of SEQ ID NO: 2. Such
fragments retain pore forming activity. Fragments may be at least
50, 100, 150 or 200 amino acids in length. Such fragments may be
used to produce the pores. A fragment preferably comprises the pore
forming domain of SEQ ID NO: 2. Fragments must include one of
residues 88, 90, 91, 105, 118 and 134 of SEQ ID NO: 2. Typically,
fragments include all of residues 88, 90, 91, 105, 118 and 134 of
SEQ ID NO: 2.
[0183] One or more amino acids may be alternatively or additionally
added to the polypeptides described above. An extension may be
provided at the amino terminal or carboxy terminal of the amino
acid sequence of SEQ ID NO: 2 or polypeptide variant or fragment
thereof. The extension may be quite short, for example from 1 to 10
amino acids in length. Alternatively, the extension may be longer,
for example up to 50 or 100 amino acids. A carrier protein may be
fused to an amino acid sequence according to the invention. Other
fusion proteins are discussed in more detail below.
[0184] As discussed above, a variant is a polypeptide that has an
amino acid sequence which varies from that of SEQ ID NO: 2 and
which retains its ability to form a pore. A variant typically
contains the regions of SEQ ID NO: 2 that are responsible for pore
formation. The pore forming ability of Msp, which contains a
.beta.-barrel, is provided by .beta.-sheets in each subunit. A
variant of SEQ ID NO: 2 typically comprises the regions in SEQ ID
NO: 2 that form .beta.-sheets. One or more modifications can be
made to the regions of SEQ ID NO: 2 that form .beta.-sheets as long
as the resulting variant retains its ability to form a pore. A
variant of SEQ ID NO: 2 preferably includes one or more
modifications, such as substitutions, additions or deletions,
within its .alpha.-helices and/or loop regions.
[0185] The monomer derived from Msp contains one or more specific
modifications to facilitate nucleotide discrimination. The monomer
derived from Msp may also contain other non-specific modifications
as long as they do not interfere with pore formation. A number of
non-specific side chain modifications are known in the art and may
be made to the side chains of the monomer derived from Msp. Such
modifications include, for example, reductive alkylation of amino
acids by reaction with an aldehyde followed by reduction with
NaBH.sub.4, amidination with methylacetimidate or acylation with
acetic anhydride.
[0186] The monomer derived from Msp can be produced using standard
methods known in the art. The monomer derived from Msp may be made
synthetically or by recombinant means. For example, the pore may be
synthesized by in vitro translation and transcription (IVTT).
Suitable methods for producing pores are discussed in International
Application Nos. PCT/GB09/001690 (published as WO 2010/004273),
PCT/GB09/001679 (published as WO 2010/004265) or PCT/GB10/000133
(published as WO 2010/086603). Methods for inserting pores into
membranes are discussed.
[0187] The transmembrane protein pore is also preferably derived
from .alpha.-hemolysin (.alpha.-HL). The wild type .alpha.-HL pore
is formed of seven identical monomers or subunits (i.e. it is
heptameric). The sequence of one monomer or subunit of
.alpha.-hemolysin-NN is shown in SEQ ID NO: 4.
[0188] In some embodiments, the transmembrane protein pore is
chemically modified. The pore can be chemically modified in any way
and at any site. The transmembrane protein pore is preferably
chemically modified by attachment of a molecule to one or more
cysteines (cysteine linkage), attachment of a molecule to one or
more lysines, attachment of a molecule to one or more non-natural
amino acids, enzyme modification of an epitope or modification of a
terminus. Suitable methods for carrying out such modifications are
well-known in the art. The transmembrane protein pore may be
chemically modified by the attachment of any molecule. For
instance, the pore may be chemically modified by attachment of a
dye or a fluorophore.
[0189] Any number of the monomers in the pore may be chemically
modified. One or more, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the
monomers is preferably chemically modified as discussed above.
[0190] The reactivity of cysteine residues may be enhanced by
modification of the adjacent residues. For instance, the basic
groups of flanking arginine, histidine or lysine residues will
change the pKa of the cysteines thiol group to that of the more
reactive S.sup.- group. The reactivity of cysteine residues may be
protected by thiol protective groups such as dTNB. These may be
reacted with one or more cysteine residues of the pore before a
linker is attached.
[0191] The molecule (with which the pore is chemically modified)
may be attached directly to the pore or attached via a linker as
disclosed in International Application Nos. PCT/GB09/001690
(published as WO 2010/004273), PCT/GB09/001679 (published as WO
2010/004265) or PCT/GB10/000133 (published as WO 2010/086603).
[0192] Any of the proteins described herein, such as the
transmembrane protein pores, can be produced using standard methods
known in the art. Polynucleotide sequences encoding a pore or
construct may be derived and replicated using standard methods in
the art. Polynucleotide sequences encoding a pore or construct may
be expressed in a bacterial host cell using standard techniques in
the art. The pore may be produced in a cell by in situ expression
of the polypeptide from a recombinant expression vector. The
expression vector optionally carries an inducible promoter to
control the expression of the polypeptide. These methods are
described in Sambrook, J. and Russell, D. (2001). Molecular
Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.
[0193] The pore may be produced in large scale following
purification by any protein liquid chromatography system from
protein producing organisms or after recombinant expression.
Typical protein liquid chromatography systems include FPLC, AKTA
systems, the Bio-Cad system, the Bio-Rad BioLogic system and the
Gilson HPLC system.
Uncoupling
[0194] The method of the invention involves uncoupling the first
analyte from the membrane. The method of the invention may involve
uncoupling the second analyte from the membrane, for instance if
three or more analytes are being investigated.
[0195] Step (c) (i.e. uncoupling of the first analyte) may be
performed before step (d) (i.e. before coupling the second analyte
to the membrane). Step (d) may be performed before step (c). If the
second analyte is coupled to the membrane before the first analyte
is uncoupled, step (c) preferably comprises selectively uncoupling
the first analyte from the membrane (i.e. uncoupling the first
analyte but not the second analyte from the membrane). A skilled
person can design a system in which selective uncoupling is
achieved. Steps (c) and (d) may be performed at the same time. This
is discussed in more detail below.
[0196] In step (c), at least 10% of the first analyte is preferably
uncoupled from the membrane. For instance, at least 20%, at least
30%, at least 40%, at least 50%, at least 60%, at least 70%, at
least 80% at least 90% or at least 95% of the first analyte may be
uncoupled from the membrane. Preferably, all of the first analyte
is uncoupled from the membrane. The amount of the first analyte
uncoupled from the membrane can be determined using the detector.
This is disclosed in the Examples.
[0197] The first analyte can be uncoupled from the membrane using
any known method. The first analyte is preferably not uncoupled
from the membrane in step (c) using the detector, such as a
transmembrane pore. The first analyte is preferably not uncoupled
from the membrane using a voltage or an applied potential.
[0198] Step (c) preferably comprises uncoupling the first analyte
from the membrane by removing the one or more anchors from the
membrane. In such embodiments, the second analyte is coupled to the
membrane using other (or separate) one or more anchors. The one or
more anchors used to couple the second analyte may be the same type
of anchor used to couple the first analyte or a different type of
anchor.
[0199] Step (c) more preferably comprises contacting the one or
more anchors with an agent which has a higher affinity for the one
or more anchors than the one or more anchors have for the membrane.
A variety of protocols for competitive binding or immunoradiometric
assays to determine the specific binding capability of molecules
are well known in the art (see for example Maddox et al, J. Exp.
Med. 158, 1211-1226, 1993). The agent removes the one or more
anchors from the membrane and thereby uncouples the first analyte.
The agent is preferably a sugar. Any sugar which binds to the one
or more anchors with a higher affinity than the one or more anchors
have for the membrane may be used. The sugar may be a cyclodextrin
or derivative thereof as discussed below.
[0200] The one or more anchors preferably comprise a hydrophobic
anchor, such as cholesterol, and the agent is preferably a
cyclodextrin or a derivative thereof or a lipid. The cyclodextrin
or derivative thereof may be any of those disclosed in Eliseev, A.
V., and Schneider, H-J. (1994) J. Am. Chem. Soc. 116, 6081-6088.
The agent is more preferably heptakis-6-amino-.beta.-cyclodextrin
(am.sub.7-.beta.CD), 6-monodeoxy-6-monoamino-.beta.-cyclodextrin
(am.sub.1-.beta.CD), heptakis-(6-deoxy-6-guanidino)-cyclodextrin
(gu.sub.7-.beta.CD),
heptakis(2,3,6-tri-O-methyl)-.beta.-cyclodextrin or
(2-hydroxypropyl)-.beta.-cyclodextrin. Any of the lipids disclosed
herein may be used.
[0201] The one or more anchors preferably comprise streptavidin,
biotin or desthiobiotin and the agent is preferably biotin,
desthiobiotin or streptavidin. Both biotin and desthiobiotin bind
to streptavidin with a higher affinity than streptavidin binds to
the membrane. Biotin has a stronger affinity for streptavidin than
desthiobiotin. An anchor comprising streptavidin may therefore be
removed from the membrane using biotin or desthiobiotin, depending
on the composition of the anchor e.g. as shown in Example 5 and
FIG. 7.
[0202] The one or more anchors preferably comprise a protein and
the agent is preferably an antibody or fragment thereof which
specifically binds to the protein. An antibody specifically binds
to a protein if it binds to the protein with preferential or high
affinity, but does not bind or binds with only low affinity to
other or different proteins. An antibody binds with preferential or
high affinity if it binds with a Kd of 1.times.10.sup.-6 M or less,
more preferably 1.times.10.sup.-7 M or less, 5.times.10.sup.-8 M or
less, more preferably 1.times.10.sup.-8 M or less or more
preferably 5.times.10.sup.-9M or less. An antibody binds with low
affinity if it binds with a Kd of 1.times.10.sup.-6 M or more, more
preferably 1.times.10.sup.-5 M or more, more preferably
1.times.10.sup.-4 M or more, more preferably 1.times.10.sup.-3 M or
more, even more preferably 1.times.10.sup.-2 M or more. Any method
may be used to detect binding or specific binding. Methods of
quantitatively measuring the binding of an antibody to a protein
are well known in the art. The antibody may be a monoclonal
antibody or a polyclonal antibody. Suitable fragments of antibodies
include, but are not limited to, Fv, F(ab') and F(ab')2 fragments,
as well as single chain antibodies. Furthermore, the antibody or
fragment thereof may be a chimeric antibody or fragment thereof, a
CDR-grafted antibody or fragment thereof or a humanised antibody or
fragment thereof.
[0203] Step (c) preferably comprises contacting the one or more
anchors with an agent which reduces their ability to couple to the
membrane. For instance, the agent could interfere with the
structure and/or hydrophobicity of the one or more anchors and
thereby reduce their ability to couple to the membrane. The one or
more anchors preferably comprise cholesterol and the agent is
preferably cholesterol dehydrogenase. The one or more anchors
preferably comprise a lipid and the agent is preferably a
phospholipase. The one or more anchors preferably comprise a
protein and the agent is preferably a proteinase or urea. Other
combination of suitable anchors and agents will be clear to a
person skilled in the art.
[0204] Step (c) preferably comprises uncoupling the first analyte
from the membrane by separating the first analyte from the one or
more anchors. This can be done in any manner. For instance, the
linker could be cut in one or more anchors comprising a linker.
This embodiment is particularly applicable to one or more anchors
which involve linkage via hybridisation. Such anchors are discussed
above.
[0205] Step (c) more preferably comprises uncoupling the first
analyte from the membrane by contacting the first analyte and the
one or more anchors with an agent which competes with the first
analyte for binding to the one or more anchors. Methods for
determining and measuring competitive binding are known in the art.
The agent is preferably a polynucleotide which competes with the
first analyte for hybridisation to the one or more anchors. For
instance, if the first analyte is coupled to the membrane using one
or more anchors which involve hybridisation, the analyte can be
uncoupled by contacting the one or more anchors with a
polynucleotide which also hybridises to the site of hybridisation.
The polynucleotide agent is typically added at a concentration that
is higher than the concentration of the first analyte and one or
more anchors. Alternatively, the polynucleotide agent may hybridise
more strongly to the one or more anchors than the first
analyte.
[0206] Step (c) more preferably comprises (i) contacting the first
analyte and the one or more anchors with urea,
tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT),
streptavidin or biotin, UV light, an enzyme or a binding agent;
(ii) heating the first analyte and one or more anchors; or (iii)
altering the pH. Urea, tris(2-carboxyethyl)phosphine (TCEP) or
dithiothreitol (DTT) are capable of disrupting anchors and
separating the first analyte from the membrane. If an anchor
comprises a streptavidin-biotin link, then a streptavidin agent
will compete for binding to the biotin. If an anchor comprises a
streptavidin-desthiobiotin link, then a biotin agent will compete
for binding to the streptavidin. UV light can be used to breakdown
photolabile groups. Enzymes and binding agents can be used to cut,
breakdown or unravel the anchor. Preferred enzymes include, but are
not limited to, an exonuclease, an endonuclease or a helicase.
Preferred binding agents include, but are not limited to, an
enzyme, an antibody or a fragment thereof or a single-stranded
binding protein (SSB). Any of the enzymes discussed below or
antibodies discussed above may be used. Heat and pH can be used to
disrupt hybridisation and other linkages.
[0207] If the first analyte is uncoupled from the membrane by
separating the first analyte from the one or more anchors, the one
or more anchors will remain in the membrane. Step (d) preferably
comprises coupling the second analyte to the membrane using the one
or more anchors that were separated from the first analyte. For
instance, the second analyte may also be provided with a
polynucleotide which hybridises to the one or more anchors that
remain in the membrane. Alternatively, step (d) preferably
comprises coupling the second analyte to the membrane using
separate one or more anchors from the ones separated from the first
analyte (i.e. other one or more anchors). The separate one or more
anchors may be the same type of anchor used to couple the first
analyte to the membrane or may be a different type of anchor. Step
(d) preferably comprises coupling the second analyte to the
membrane using a different one or more anchors from the ones
separated from the first analyte.
[0208] In a preferred embodiment, steps (c) and (d) comprise
uncoupling the first analyte from the membrane by contacting the
membrane with the second analyte such that the second analyte
competes with the first analyte for binding to the one or more
anchors and replaces the first analyte. For instance, if the first
analyte is coupled to the membrane using one or more anchors which
involve hybridisation, the analyte can be uncoupled by contacting
the one or more anchors with the second analyte attached to a
polynucleotide which also hybridises to the sites of hybridisation
in the one or more anchors. The second analyte is typically added
at a concentration that is higher than the concentration of the
first analyte and one or more anchors. Alternatively, the second
analyte may hybridise more strongly to the one or more anchors than
the first analyte.
Removal or Washing
[0209] Although the first analyte is uncoupled from the membrane in
step (c), it is not necessarily removed or washed away. If the
second analyte can be easily distinguished from the first analyte,
there is no need to remove the first analyte.
[0210] Between steps (c) and (d), the method preferably further
comprises removing at least some of the first sample from the
membrane. At least 10% of the first sample may be removed, such as
at least 20%, at least 30%, at least 40%, at least 50%, at least
60%, at least 70%, at least 80% or at least 90% of the first sample
may be removed. The method more preferably further comprises
removing all of the first sample from the membrane. This can be
done in any way. For instance, the membrane can be washed with a
buffer after the first analyte has been uncoupled. Suitable buffers
are discussed below.
Polynucleotide Characterisation
[0211] The method of the invention preferably involves measuring
one or more characteristics of two or more polynucleotides. The two
or more polynucleotides may be different polynucleotides or two
instances of the same polynucleotide.
[0212] Any number of polynucleotides can be investigated. For
instance, the method of the invention may concern determining the
presence, absence or one or more characteristics of 3, 4, 5, 6, 7,
8, 9, 10, 20, 30, 50, 100 or more polynucleotides. If three or more
polynucleotides are investigated using the method of the invention,
the second polynucleotide is also uncoupled from the membrane and
the requisite number of steps are added for the third
polynucleotide. The same is true for four or more
polynucleotides.
[0213] The polynucleotides can be naturally occurring or
artificial. For instance, the method may be used to verify the
sequence of two or more manufactured oligonucleotides. The methods
are typically carried out in vitro.
[0214] The method may involve measuring two, three, four or five or
more characteristics of each polynucleotide. The one or more
characteristics are preferably selected from (i) the length of the
polynucleotide, (ii) the identity of the polynucleotide, (iii) the
sequence of the polynucleotide, (iv) the secondary structure of the
polynucleotide and (v) whether or not the polynucleotide is
modified. Any combination of (i) to (v) may be measured in
accordance with the invention, such as {i}, {ii}, {iii}, {iv}, {v},
{i,ii}, {i,iii}, {i,iv}, {i,v}, {ii,iii}, {ii,iv}, {ii,v},
{iii,iv}, {iii,v}, {iv,v}, {i,ii,iii}, {i,ii,iv}, {i,ii,v},
{i,iii,iv}, {i,iii,v}, {i,iv,v}, {ii,iii,iv}, {ii,iii,v},
{ii,iv,v}, {iii,iv,v}, {i,ii,iii,iv}, {i,ii,iii,v}, {i,ii,iv,v},
{i,iii,iv,v}, {ii,iii,iv,v} or {i,ii,iii,iv,v}. Different
combinations of (i) to (v) may be measured for the first
polynucleotide compared with the second polynucleotide, including
any of those combinations listed above.
[0215] For (i), the length of the polynucleotide may be measured
for example by determining the number of interactions between the
polynucleotide and the pore or the duration of interaction between
the polynucleotide and the pore.
[0216] For (ii), the identity of the polynucleotide may be measured
in a number of ways. The identity of the polynucleotide may be
measured in conjunction with measurement of the sequence of the
polynucleotide or without measurement of the sequence of the
polynucleotide. The former is straightforward; the polynucleotide
is sequenced and thereby identified. The latter may be done in
several ways. For instance, the presence of a particular motif in
the polynucleotide may be measured (without measuring the remaining
sequence of the polynucleotide). Alternatively, the measurement of
a particular electrical and/or optical signal in the method may
identify the polynucleotide as coming from a particular source.
[0217] For (iii), the sequence of the polynucleotide can be
determined as described previously. Suitable sequencing methods,
particularly those using electrical measurements, are described in
Stoddart D et al., Proc Natl Acad Sci, 12; 106(19):7702-7,
Lieberman K R et al, J Am Chem Soc. 2010; 132(50):17961-72, and
International Application WO 2000/28312.
[0218] For (iv), the secondary structure may be measured in a
variety of ways. For instance, if the method involves an electrical
measurement, the secondary structure may be measured using a change
in dwell time or a change in current flowing through the pore. This
allows regions of single-stranded and double-stranded
polynucleotide to be distinguished.
[0219] For (v), the presence or absence of any modification may be
measured. The method preferably comprises determining whether or
not the polynucleotide is modified by methylation, by oxidation, by
damage, with one or more proteins or with one or more labels, tags
or spacers. Specific modifications will result in specific
interactions with the pore which can be measured using the methods
described below. For instance, methylcytosine may be distinguished
from cytosine on the basis of the current flowing through the pore
during its interaction with each nucleotide.
[0220] The methods may be carried out using any apparatus that is
suitable for investigating a membrane/pore system in which a pore
is present in a membrane. The method may be carried out using any
apparatus that is suitable for transmembrane pore sensing. For
example, the apparatus comprises a chamber comprising an aqueous
solution and a barrier that separates the chamber into two
sections. The barrier typically has an aperture in which the
membrane containing the pore is formed. Alternatively the barrier
forms the membrane in which the pore is present.
[0221] The methods may be carried out using the apparatus described
in International Application No. PCT/GB08/000562 (WO
2008/102120).
[0222] The methods may involve measuring the current passing
through the pore as the polynucleotide moves with respect to the
pore. Therefore the apparatus may also comprise an electrical
circuit capable of applying a potential and measuring an electrical
signal across the membrane and pore. The methods may be carried out
using a patch clamp or a voltage clamp. The methods preferably
involve the use of a voltage clamp.
[0223] The methods of the invention may involve the measuring of a
current passing through the pore as the polynucleotide moves with
respect to the pore. Suitable conditions for measuring ionic
currents through transmembrane protein pores are known in the art
and disclosed in the Example. The method is typically carried out
with a voltage applied across the membrane and pore. The voltage
used is typically from +5 V to -5 V, such as from +4 V to -4 V, +3
V to -3 V or +2 V to -2 V. The voltage used is typically from -600
mV to +600 mV or -400 mV to +400 mV. The voltage used is preferably
in a range having a lower limit selected from -400 mV, -300 mV,
-200 mV, -150 mV, -100 mV, -50 mV, -20 mV and 0 mV and an upper
limit independently selected from +10 mV, +20 mV, +50 mV, +100 mV,
+150 mV, +200 mV, +300 mV and +400 mV. The voltage used is more
preferably in the range 100 mV to 240 mV and most preferably in the
range of 120 mV to 220 mV. It is possible to increase
discrimination between different nucleotides by a pore by using an
increased applied potential.
[0224] The methods are typically carried out in the presence of any
charge carriers, such as metal salts, for example alkali metal
salt, halide salts, for example chloride salts, such as alkali
metal chloride salt. Charge carriers may include ionic liquids or
organic salts, for example tetramethyl ammonium chloride,
trimethylphenyl ammonium chloride, phenyltrimethyl ammonium
chloride, or 1-ethyl-3-methyl imidazolium chloride. In the
exemplary apparatus discussed above, the salt is present in the
aqueous solution in the chamber. Potassium chloride (KCl), sodium
chloride (NaCl), caesium chloride (CsCl) or a mixture of potassium
ferrocyanide and potassium ferricyanide is typically used. KCl,
NaCl and a mixture of potassium ferrocyanide and potassium
ferricyanide are preferred. The charge carriers may be asymmetric
across the membrane. For instance, the type and/or concentration of
the charge carriers may be different on each side of the
membrane.
[0225] The salt concentration may be at saturation. The salt
concentration may be 3 M or lower and is typically from 0.1 to 2.5
M, from 0.3 to 1.9 M, from 0.5 to 1.8 M, from 0.7 to 1.7 M, from
0.9 to 1.6 M or from 1 M to 1.4 M. The salt concentration is
preferably from 150 mM to 1 M. The method is preferably carried out
using a salt concentration of at least 0.3 M, such as at least 0.4
M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M,
at least 1.5 M, at least 2.0 M, at least 2.5 M or at least 3.0 M.
High salt concentrations provide a high signal to noise ratio and
allow for currents indicative of the presence of a nucleotide to be
identified against the background of normal current
fluctuations.
[0226] The methods are typically carried out in the presence of a
buffer. In the exemplary apparatus discussed above, the buffer is
present in the aqueous solution in the chamber. Any buffer may be
used in the method of the invention. Typically, the buffer is
phosphate buffer. Other suitable buffers are HEPES and Tris-HCl
buffer. The methods are typically carried out at a pH of from 4.0
to 12.0, from 4.5 to 10.0, from 5.0 to 9.0, from 5.5 to 8.8, from
6.0 to 8.7 or from 7.0 to 8.8 or 7.5 to 8.5. The pH used is
preferably about 7.5.
[0227] The methods may be carried out at from 0.degree. C. to
100.degree. C., from 15.degree. C. to 95.degree. C., from
16.degree. C. to 90.degree. C., from 17.degree. C. to 85.degree.
C., from 18.degree. C. to 80.degree. C., 19.degree. C. to
70.degree. C., or from 20.degree. C. to 60.degree. C. The methods
are typically carried out at room temperature. The methods are
optionally carried out at a temperature that supports enzyme
function, such as about 37.degree. C.
[0228] Step (b) preferably comprises allowing the first
polynucleotide to interact with a polynucleotide binding protein
which controls the interaction of the first polynucleotide with the
detector present in the membrane and/or step (e) preferably
comprises allowing the second polynucleotide to interact with a
polynucleotide binding protein which controls the interaction of
the second polynucleotide with the detector present in the
membrane.
[0229] More preferably, the method comprises (a) coupling a first
polynucleotide in a first sample to a membrane using one or more
anchors; (b) contacting the first polynucleotide with a
transmembrane pore such that the first polynucleotide moves through
the pore; (c) taking one or more measurements as the first
polynucleotide moves with respect to the pore wherein the
measurements are indicative of one or more characteristics of the
first polynucleotide and thereby characterising the first
polynucleotide; (d) uncoupling the first polynucleotide from the
membrane; (e) coupling a second polynucleotide in a second sample
to the membrane using one or more anchors; (f) contacting the
second polynucleotide with a transmembrane pore such that the
second polynucleotide moves through the pore; and (g) taking one or
more measurements as the second polynucleotide moves with respect
to the pore wherein the measurements are indicative of one or more
characteristics of the second polynucleotide and thereby
characterising the second polynucleotide. In this embodiment, step
(b) preferably comprises contacting the first polynucleotide with a
transmembrane pore and a polynucleotide binding protein such that
the protein controls the movement of the first polynucleotide
through the pore and/or step (f) preferably comprises contacting
the second polynucleotide with a transmembrane pore and a
polynucleotide binding protein such that the protein controls the
movement of the second polynucleotide through the pore.
[0230] The polynucleotide binding protein may be any protein that
is capable of binding to the polynucleotide and controlling its
movement through the pore. It is straightforward in the art to
determine whether or not a protein binds to a polynucleotide. The
protein typically interacts with and modifies at least one property
of the polynucleotide. The protein may modify the polynucleotide by
cleaving it to form individual nucleotides or shorter chains of
nucleotides, such as di- or trinucleotides. The moiety may modify
the polynucleotide by orienting it or moving it to a specific
position, i.e. controlling its movement.
[0231] The polynucleotide binding protein is preferably derived
from a polynucleotide handling enzyme. A polynucleotide handling
enzyme is a polypeptide that is capable of interacting with and
modifying at least one property of a polynucleotide. The enzyme may
modify the polynucleotide by cleaving it to form individual
nucleotides or shorter chains of nucleotides, such as di- or
trinucleotides. The enzyme may modify the polynucleotide by
orienting it or moving it to a specific position. The
polynucleotide handling enzyme does not need to display enzymatic
activity as long as it is capable of binding the polynucleotide and
controlling its movement through the pore. For instance, the enzyme
may be modified to remove its enzymatic activity or may be used
under conditions which prevent it from acting as an enzyme. Such
conditions are discussed in more detail below.
[0232] The polynucleotide handling enzyme is preferably derived
from a nucleolytic enzyme. The polynucleotide handling enzyme used
in the construct of the enzyme is more preferably derived from a
member of any of the Enzyme Classification (EC) groups 3.1.11,
3.1.13, 3.1.14, 3.1.15, 3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26,
3.1.27, 3.1.30 and 3.1.31. The enzyme may be any of those disclosed
in International Application No. PCT/GB10/000133 (published as WO
2010/086603).
[0233] Preferred enzymes are polymerases, exonucleases, helicases
and topoisomerases, such as gyrases. Suitable enzymes include, but
are not limited to, exonuclease I from E. coli (SEQ ID NO: 11),
exonuclease III enzyme from E. coli (SEQ ID NO: 13), RecJ from T.
thermophilus (SEQ ID NO: 15) and bacteriophage lambda exonuclease
(SEQ ID NO: 17) and variants thereof. Three subunits comprising the
sequence shown in SEQ ID NO: 15 or a variant thereof interact to
form a trimer exonuclease. The enzyme is preferably Phi29 DNA
polymerase (SEQ ID NO: 9) or a variant thereof. The topoisomerase
is preferably a member of any of the Moiety Classification (EC)
groups 5.99.1.2 and 5.99.1.3.
[0234] The enzyme is most preferably derived from a helicase, such
as Hel308 Mbu (SEQ ID NO: 18), Hel308 Csy (SEQ ID NO: 19), Hel308
Mhu (SEQ ID NO: 20), TraI Eco (SEQ ID NO: 21), XPD Mbu (SEQ ID NO:
22) or a variant thereof. Any helicase may be used in the
invention. The helicase may be or be derived from a Hel308
helicase, a RecD helicase, such as TraI helicase or a TrwC
helicase, a XPD helicase or a Dda helicase. The helicase may be any
of the helicases, modified helicases or helicase constructs
disclosed in International Application Nos. PCT/GB2012/052579
(published as WO 2013/057495); PCT/GB2012/053274 (published as WO
2013/098562); PCT/GB2012/053273 (published as WO2013098561);
PCT/GB2013/051925 (published as WO 2014/013260); PCT/GB2013/051924
(published as WO 2014/013259); PCT/GB2013/051928 (published as WO
2014/013262) and PCT/GB2014/052736.
[0235] The helicase preferably comprises the sequence shown in SEQ
ID NO: 25 (Trwc Cba) or a variant thereof, the sequence shown in
SEQ ID NO: 18 (Hel308 Mbu) or a variant thereof or the sequence
shown in SEQ ID NO: 24 (Dda) or a variant thereof. Variants may
differ from the native sequences in any of the ways discussed below
for transmembrane pores. A preferred variant of SEQ ID NO: 24
comprises E94C/A360C and then (.DELTA.M1)G1G2 (i.e. deletion of M1
and then addition G1 and G2) or E94C/A360C/C109A/C136A and then
(.DELTA.M1)G1G2.
[0236] Any number of helicases may be used in accordance with the
invention. For instance, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
helicases may be used. In some embodiments, different numbers of
helicases may be used.
[0237] The method of the invention preferably comprises contacting
the first polynucleotide analyte and/or the second polynucleotide
analyte with two or more helicases. The two or more helicases are
typically the same helicase. The two or more helicases may be
different helicases.
[0238] The two or more helicases may be any combination of the
helicases mentioned above. The two or more helicases may be two or
more Dda helicases. The two or more helicases may be one or more
Dda helicases and one or more TrwC helicases. The two or more
helicases may be different variants of the same helicase.
[0239] The two or more helicases are preferably attached to one
another. The two or more helicases are more preferably covalently
attached to one another. The helicases may be attached in any order
and using any method. Preferred helicase constructs for use in the
invention are described in International Application Nos.
PCT/GB2013/051925 (published as WO 2014/013260); PCT/GB2013/051924
(published as WO 2014/013259); PCT/GB2013/051928 (published as WO
2014/013262) and PCT/GB2014/052736.
[0240] A variant of SEQ ID NOs: 9, 11, 13, 15, 17, 18, 19, 20, 21,
22, 23, 24 or 25 is an enzyme that has an amino acid sequence which
varies from that of SEQ ID NO: 9, 11, 13, 15, 17, 18, 19, 20, 21,
22, 23, 24 or 25 and which retains polynucleotide binding ability.
This can be measured using any method known in the art. For
instance, the variant can be contacted with a polynucleotide and
its ability to bind to and move along the polynucleotide can be
measured. The variant may include modifications that facilitate
binding of the polynucleotide and/or facilitate its activity at
high salt concentrations and/or room temperature. Variants may be
modified such that they bind polynucleotides (i.e. retain
polynucleotide binding ability) but do not function as a helicase
(i.e. do not move along polynucleotides when provided with all the
necessary components to facilitate movement, e.g. ATP and
Mg.sup.2+). Such modifications are known in the art. For instance,
modification of the Mg.sup.2+ binding domain in helicases typically
results in variants which do not function as helicases. These types
of variants may act as molecular brakes (see below).
[0241] Over the entire length of the amino acid sequence of SEQ ID
NO: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25, a variant
will preferably be at least 50% homologous to that sequence based
on amino acid identity. More preferably, the variant polypeptide
may be at least 55%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90% and more
preferably at least 95%, 97% or 99% homologous based on amino acid
identity to the amino acid sequence of SEQ ID NO: 9, 11, 13, 15,
17, 18, 19, 20, 21, 22, 23, 24 or 25 over the entire sequence.
There may be at least 80%, for example at least 85%, 90% or 95%,
amino acid identity over a stretch of 200 or more, for example 230,
250, 270, 280, 300, 400, 500, 600, 700, 800, 900 or 1000 or more,
contiguous amino acids ("hard homology"). Homology is determined as
described above. The variant may differ from the wild-type sequence
in any of the ways discussed above with reference to SEQ ID NO: 2
and 4 above. The enzyme may be covalently attached to the pore. Any
method may be used to covalently attach the enzyme to the pore.
[0242] A preferred molecular brake is TrwC Cba-Q594A (SEQ ID NO: 25
with the mutation Q594A). This variant does not function as a
helicase (i.e. binds polynucleotides but does not move along them
when provided with all the necessary components to facilitate
movement, e.g. ATP and Mg.sup.2+).
[0243] In strand sequencing, the polynucleotide is translocated
through the pore either with or against an applied potential.
Exonucleases that act progressively or processively on double
stranded polynucleotides can be used on the cis side of the pore to
feed the remaining single strand through under an applied potential
or the trans side under a reverse potential. Likewise, a helicase
that unwinds the double stranded DNA can also be used in a similar
manner. A polymerase may also be used. There are also possibilities
for sequencing applications that require strand translocation
against an applied potential, but the DNA must be first "caught" by
the enzyme under a reverse or no potential. With the potential then
switched back following binding the strand will pass cis to trans
through the pore and be held in an extended conformation by the
current flow. The single strand DNA exonucleases or single strand
DNA dependent polymerases can act as molecular motors to pull the
recently translocated single strand back through the pore in a
controlled stepwise manner, trans to cis, against the applied
potential.
Helicase(s) and Molecular Brake(s)
[0244] In a preferred embodiment, the method comprises: [0245] (a)
providing a first polynucleotide in a first sample with one or more
helicases attached to the first polynucleotide and one or more
molecular brakes attached to the first polynucleotide; [0246] (b)
providing a second polynucleotide in a second sample with one or
more helicases attached to the second polynucleotide and one or
more molecular brakes attached to the second polynucleotide; [0247]
(c) coupling the first polynucleotide in the first sample to a
membrane using one or more anchors; [0248] (d) contacting the first
polynucleotide with a transmembrane pore and applying a potential
across the pore such that the one or more helicases and the one or
more molecular brakes are brought together and both control the
movement of the first polynucleotide through the pore; [0249] (e)
taking one or more measurements as the first polynucleotide moves
with respect to the pore wherein the measurements are indicative of
one or more characteristics of the first polynucleotide and thereby
characterising the first polynucleotide; [0250] (f) uncoupling the
first polynucleotide from the membrane; [0251] (g) coupling the
second polynucleotide in the second sample to the membrane using
one or more anchors; [0252] (h) contacting the second
polynucleotide with a transmembrane pore and applying a potential
across the pore such that the one or more helicases and the one or
more molecular brakes are brought together and both control the
movement of the second polynucleotide through the pore; and [0253]
(i) taking one or more measurements as the second polynucleotide
moves with respect to the pore wherein the measurements are
indicative of one or more characteristics of the second
polynucleotide and thereby characterising the second
polynucleotide.
[0254] This type of method is discussed in detail in the
International Application PCT/GB2014/052737.
[0255] Step (f) (i.e. uncoupling of the first polynucleotide) may
be performed before step (g) (i.e. before coupling the second
polynucleotide to the membrane). Step (g) may be performed before
step (f). If the second polynucleotide is coupled to the membrane
before the first polynucleotide is uncoupled, step (f) preferably
comprises selectively uncoupling the first polynucleotide from the
membrane (i.e. uncoupling the first polynucleotide but not the
second polynucleotide from the membrane). A skilled person can
design a system in which selective uncoupling is achieved. Steps
(f) and (g) may be performed at the same time. This is discussed in
more detail below.
[0256] The one or more helicases may be any of those discussed
above. The one or more molecular brakes may be any compound or
molecule which binds to the polynucleotide and slow the movement of
the polynucleotide through the pore. The one or more molecular
brakes are preferably one or more polynucleotide binding proteins.
The polynucleotide binding protein may be any protein that is
capable of binding to the polynucleotide and controlling its
movement through the pore. It is straightforward in the art to
determine whether or not a protein binds to a polynucleotide. The
protein typically interacts with and modifies at least one property
of the polynucleotide. The protein may modify the polynucleotide by
cleaving it to form individual nucleotides or shorter chains of
nucleotides, such as di- or trinucleotides. The moiety may modify
the polynucleotide by orienting it or moving it to a specific
position, i.e. controlling its movement.
[0257] The polynucleotide binding protein is preferably derived
from a polynucleotide handling enzyme. The one or more molecular
brakes may be derived from any of the polynucleotide handling
enzymes discussed above. Modified versions of Phi29 polymerase (SEQ
ID NO: 8) which act as molecular brakes are disclosed in U.S. Pat.
No. 5,576,204. The one or more molecular brakes are preferably
derived from a helicase.
Spacers in Polynucleotide Analytes
[0258] The one or more helicases may be stalled at one or more
spacers as discussed in International Application No.
PCT/GB2014/050175 (published as WO 2014/135838). Any configuration
of one or more helicases and one or more spacers disclosed in the
International Application may be used in this invention.
Double Stranded Polynucleotide
[0259] The first polynucleotide analyte and/or the second
polynucleotide analyte may be double stranded. If the analyte
polynucleotide is double stranded, the method preferably further
comprises before the coupling step ligating a hairpin adaptor to
one end of the polynucleotide and separating the two strands of the
polynucleotide to form a single stranded polynucleotide construct.
The single stranded polynucleotide construct may then be allowed to
interact with the detector in accordance with the invention.
Linking and interrogating both strands on a double stranded
construct in this way increases the efficiency and accuracy of
characterization. Sequencing using hairpin adaptors is disclosed in
International Application Nos. PCT/GB2010/000160 (published as WO
2010/086622) and PCT/GB2012/051786 (published as WO
2013/014451).
Leader Sequence
[0260] Before the coupling step, the method preferably comprises
attaching to the first and/or second polynucleotide analyte a
leader sequence which preferentially threads into the pore. The
leader sequence facilitates the method of the invention. The leader
sequence is designed to preferentially thread into the
transmembrane pore and thereby facilitate the movement of
polynucleotide analyte through the pore. The leader sequence can
also be used to link the polynucleotide to the one or more anchors
as discussed above.
[0261] The leader sequence typically comprises a polymer. The
polymer is preferably negatively charged. The polymer is preferably
a polynucleotide, such as DNA or RNA, a modified polynucleotide
(such as abasic DNA), PNA, LNA, polyethylene glycol (PEG) or a
polypeptide. The leader preferably comprises a polynucleotide and
more preferably comprises a single stranded polynucleotide. The
leader sequence can comprise any of the polynucleotides discussed
above. The single stranded leader sequence most preferably
comprises a single strand of DNA, such as a poly dT section. The
leader sequence preferably comprises the one or more spacers.
[0262] The leader sequence can be any length, but is typically 10
to 150 nucleotides in length, such as from 20 to 150 nucleotides in
length. The length of the leader typically depends on the
transmembrane pore used in the method.
Double Coupling
[0263] The method of the invention may involve double coupling of
multiple double stranded polynucleotides. In a preferred
embodiment, the invention involves characterising multiple double
stranded polynucleotides. The method preferably comprises: [0264]
(a) providing a first double stranded polynucleotide in a first
sample with a Y adaptor at one end and a hairpin loop adaptor at
the other end, wherein the Y adaptor comprises one or more first
anchors for coupling the polynucleotide to the membrane, wherein
the hairpin loop adaptor comprises one or more second anchors for
coupling the polynucleotide to the membrane and wherein the
strength of coupling of the hairpin loop adaptor to the membrane is
greater than the strength of coupling of the Y adaptor to the
membrane; [0265] (b) providing a second double stranded
polynucleotide in a second sample in a form as defined in step (a);
[0266] (c) coupling the first polynucleotide provided in step (a)
to a membrane; [0267] (d) contacting the first polynucleotide
coupled in step (c) with a transmembrane pore such that at least
one strand of the first polynucleotide moves through the pore;
[0268] (e) taking one or more measurements as the at least one
strand of the first polynucleotide moves with respect to the pore
wherein the measurements are indicative of one or more
characteristics of the at least one strand of the first
polynucleotide and thereby characterising the first polynucleotide;
[0269] (f) uncoupling the first polynucleotide from the membrane;
[0270] (g) coupling the second polynucleotide provided in step (b)
to the membrane; [0271] (h) contacting the second polynucleotide
coupled in step (g) with a transmembrane pore such that at least
one strand of the second polynucleotide moves through the pore; and
[0272] (i) taking one or more measurements as the at least one
strand of the second polynucleotide moves with respect to the pore
wherein the measurements are indicative of one or more
characteristics of the at least one strand of the second
polynucleotide and thereby characterising the first
polynucleotide.
[0273] This type of method is discussed in detail in the UK
Applications 1406147.7 and 1407815.8 and in the International
application being filed concurrently with this application.
[0274] The double stranded polynucleotide is provided with a Y
adaptor at one end and a hairpin loop adaptor at the other end. The
Y adaptor and/or the hairpin adaptor are typically polynucleotide
adaptors. They may be formed from any of the polynucleotides
discussed above.
[0275] The Y adaptor typically comprises (a) a double stranded
region and (b) a single stranded region or a region that is not
complementary at the other end. The Y adaptor may be described as
having an overhang if it comprises a single stranded region. The
presence of a non-complementary region in the Y adaptor gives the
adaptor its Y shape since the two strands typically do not
hybridise to each other unlike the double stranded portion. The Y
adaptor comprises the one or more first anchors. Anchors are
discussed in more detail above.
[0276] The Y adaptor preferably comprises a leader sequence which
preferentially threads into the pore. Leader sequences are
discussed above.
[0277] The hairpin adaptor preferably comprises a selectable
binding moiety as discussed above. The hairpin adaptor and/or the
selectable binding moiety may comprise a region that can be cut,
nicked, cleaved or hydrolysed as discussed above.
[0278] The Y adaptor and/or the hairpin adaptor may be ligated to
the polynucleotide using any method known in the art. One or both
of the adaptors may be ligated using a ligase, such as T4 DNA
ligase, E. coli DNA ligase, Taq DNA ligase, Tma DNA ligase and
9.degree. N DNA ligase. Alternatively, the adaptors may be added to
the polynucleotide using the methods of the invention discussed
below.
[0279] In a preferred embodiment, step a) of the method comprises
modifying the double stranded polynucleotide so that it comprises
the Y adaptor at one end and the hairpin loop adaptor at the other
end. Any manner of modification can be used. The method preferably
comprises modifying the double stranded polynucleotide in
accordance with the invention. This is discussed in more detail
below. The methods of modification and characterisation may be
combined in any way.
[0280] The strength of coupling (or binding) of the hairpin adaptor
to the membrane is greater than the strength of coupling (or
binding) of the Y adaptor to the membrane. This can be measured in
any way. A suitable method for measuring the strength of coupling
(or binding) is disclosed in the Examples of the UK Applications
1406147.7 and 1407815.8 and in the International application which
is being filed concurrently.
[0281] The strength of coupling (or binding) of the hairpin loop
adaptor is preferably at least 1.5 times the strength of coupling
(or binding) of the hairpin loop adaptor, such as at least twice,
at least three times, at least four times, at least five or at
least ten times the strength of coupling (or binding) of the anchor
adaptor. The affinity constant (Kd) of the hairpin loop adaptor for
the membrane is preferably at least 1.5 times the affinity constant
of the Y adaptor, such as at least twice, at least three times, at
least four times, at least five or at least ten times the strength
of coupling of the Y adaptor.
[0282] There are several ways in which the hairpin loop adaptor
couples (or binds) more strongly to the membrane than the Y
adaptor. For instance, the hairpin loop adaptor may comprise more
anchors that than the Y adaptor. For instance, the hairpin loop
adaptor may comprise 2, 3 or more second anchors whereas the Y
adaptor may comprise one first anchor.
[0283] The strength of coupling (or binding) of the one or more
second anchors to the membrane may be greater than the strength of
coupling (or binding) of the one or more first anchors to the
membrane. The strength of coupling (or binding) of the one or more
second anchors to the hairpin loop adaptor may be greater than the
strength of coupling (or binding) of the one or more first anchors
to the Y adaptor. The one or more first anchors and the one or more
second anchors may be attached to their respective adaptors via
hybridisation and the strength of hybridisation is greater in the
one or more second anchors than in the one or more first anchors.
Any combination of these embodiments may also be used in the
invention. Strength of coupling (or binding) may be measure using
known techniques in the art.
[0284] The one or more second anchors preferably comprise one or
more groups which couples(s) (or bind(s)) to the membrane with a
greater strength than the one or more groups in the one or more
first anchors which couple(s) (or bind(s)) to the membrane. In
preferred embodiments, the hairpin loop adaptor/one or more second
anchors couple (or bind) to the membrane using cholesterol and the
Y adaptor/one or more first anchors couple (or bind) to the
membrane using palmitate. Cholesterol binds to triblock copolymer
membranes and lipid membranes more strongly than palmitate. In an
alternative embodiment, the hairpin loop adaptor/one or more second
anchors couple (or bind) to the membrane using a mono-acyl species,
such as palmitate, and the Y adaptor/one or more first anchors
couple (or bind) to the membrane using a diacyl species, such as
dipalmitoylphosphatidylcholine.
Adding Hairpin Loops and Leader Sequences
[0285] Before the coupling step, a double stranded polynucleotide
analyte is preferably contacted with a MuA transposase and a
population of double stranded MuA substrates, wherein a proportion
of the substrates in the population are Y adaptors comprising the
leader sequence and wherein a proportion of the substrates in the
population are hairpin loop adaptors. The transposase fragments the
double stranded polynucleotide analyte and ligates MuA substrates
to one or both ends of the fragments. This produces a plurality of
modified double stranded polynucleotides comprising the leader
sequence at one end and the hairpin loop at the other. The modified
double stranded polynucleotides may then be investigated using the
method of the invention.
[0286] These MuA based methods are disclosed in the International
Application No. PCT/GB2014/052505 (published as WO2015022544). They
are also discussed in detail in the UK Applications 1406147.7 and
1407815.8 and the International application being filed
concurrently with this application (ONT IP 056).
Modified Polynucleotide Analytes
[0287] Before characterisation, the first polynucleotide analyte
and/or the second polynucleotide analyte may modified by contacting
the polynucleotide analyte with a polymerase and a population of
free nucleotides under conditions in which the polymerase forms a
modified polynucleotide analyte using the polynucleotide analyte as
a template, wherein the polymerase replaces one or more of the
nucleotide species in the polynucleotide analyte with a different
nucleotide species when forming the modified polynucleotide
analyte. The modified polynucleotide analyte may then be coupled to
the membrane as in step a) and/or d). This type of modification is
described in International Application No. PCT/GB2015/050483. Any
of the polymerases discussed above may be used. The polymerase is
preferably Klenow or 90 North.
[0288] The template polynucleotide is contacted with the polymerase
under conditions in which the polymerase forms a modified
polynucleotide using the template polynucleotide as a template.
Such conditions are known in the art. For instance, the
polynucleotide is typically contacted with the polymerase in
commercially available polymerase buffer, such as buffer from New
England Biolabs.RTM.. The temperature is preferably from 20 to
37.degree. C. for Klenow or from 60 to 75.degree. C. for 9o North.
A primer or a 3' hairpin is typically used as the nucleation point
for polymerase extension.
[0289] Characterisation, such as sequencing, of a polynucleotide
using a transmembrane pore typically involves analyzing polymer
units made up of k nucleotides where k is a positive integer (i.e.
`k-mers`). This is discussed in International Application No.
PCT/GB2012/052343 (published as WO 2013/041878). While it is
desirable to have clear separation between current measurements for
different k-mers, it is common for some of these measurements to
overlap. Especially with high numbers of polymer units in the
k-mer, i.e. high values of k, it can become difficult to resolve
the measurements produced by different k-mers, to the detriment of
deriving information about the polynucleotide, for example an
estimate of the underlying sequence of the polynucleotide.
[0290] By replacing one or more nucleotide species in the template
polynucleotide analyte with different nucleotide species in the
modified polynucleotide analyte, the modified polynucleotide
analyte contains k-mers which differ from those in the template
polynucleotide analyte. The different k-mers in the modified
polynucleotide analyte are capable of producing different current
measurements from the k-mers in the template polynucleotide analyte
and so the modified polynucleotide analyte provides different
information from the template polynucleotide analyte. The
additional information from the modified polynucleotide analyte can
make it easier to characterise the template polynucleotide analyte.
In some instances, the modified polynucleotide analyte itself may
be easier to characterise. For instance, the modified
polynucleotide may be designed to include k-mers with an increased
separation or a clear separation between their current measurements
or k-mers which have a decreased noise.
Preferred Embodiment
[0291] The invention provides a method of characterising two or
more double stranded polynucleotides, comprising [0292] (a)
providing a first double stranded polynucleotide in a first sample
with a first Y adaptor at one end and a first hairpin loop adaptor
at the other end, wherein the first Y adaptor comprises one or more
first helicases and one or more first anchors for coupling the
polynucleotide to the membrane, wherein the first hairpin loop
adaptor comprises the one or more first molecular brakes and one or
more second anchors for coupling the first polynucleotide to the
membrane and wherein the strength of coupling of the first hairpin
loop adaptor to the membrane is greater than the strength of
coupling of the first Y adaptor to the membrane; [0293] (b)
providing a second double stranded polynucleotide in a second
sample with a second Y adaptor at one end and a second hairpin loop
adaptor at the other end, wherein the second Y adaptor comprises
one or more second helicases and one or more third anchors for
coupling the polynucleotide to the membrane, wherein the second
hairpin loop adaptor comprises one or more second molecular brakes
and one or more fourth anchors for coupling the second
polynucleotide to the membrane and wherein the strength of coupling
of the second hairpin loop adaptor to the membrane is greater than
the strength of coupling of the second Y adaptor to the membrane;
[0294] (c) coupling the first polynucleotide in the first sample to
a membrane; [0295] (d) contacting the first polynucleotide with a
transmembrane pore and applying a potential across the pore such
that the one or more helicases and the one or more molecular brakes
are brought together and both control the movement of the first
polynucleotide through the pore; [0296] (e) taking one or more
measurements as the first polynucleotide moves with respect to the
pore wherein the measurements are indicative of one or more
characteristics of the first polynucleotide and thereby
characterising the first polynucleotide; [0297] (f) uncoupling the
first polynucleotide from the membrane; [0298] (g) coupling the
second polynucleotide in the second sample to the membrane; [0299]
(h) contacting the second polynucleotide with a transmembrane pore
and applying a potential across the pore such that the one or more
helicases and the one or more molecular brakes are brought together
and both control the movement of the second polynucleotide through
the pore; and [0300] (i) taking one or more measurements as the
second polynucleotide moves with respect to the pore wherein the
measurements are indicative of one or more characteristics of the
second polynucleotide and thereby characterising the second
polynucleotide [0301] This combines the methods disclosed in the UK
Applications 1406155.0, 1406147.7, 1407815.8 and 1406151.9 and
International Application PCT/GB2014/052737 and the International
application being co-filed at this time (ONT IP 056). Any of the
embodiments disclosed herein and therein may be applied to the
preferred embodiment.
Other Characterisation Method
[0302] In another embodiment, a first polynucleotide analyte and/or
a second polynucleotide analyte is characterised by detecting
labelled species that are released as a polymerase incorporates
nucleotides into the polynucleotide. The polymerase uses the first
and/or second polynucleotide analyte as a template. Each labelled
species is specific for each nucleotide. The first and/or second
polynucleotide analyte is contacted with a transmembrane pore, a
polymerase and labelled nucleotides such that phosphate labelled
species are sequentially released when nucleotides are added to the
polynucleotide(s) by the polymerase, wherein the phosphate species
contain a label specific for each nucleotide. The polymerase may be
any of those discussed above. The phosphate labelled species are
detected using the pore and thereby characterising the first and/or
second polynucleotide analyte. This type of method is disclosed in
European Application No. 13187149.3 (published as EP 2682460). Any
of the embodiments discussed above equally apply to this
method.
Method Involving Cholesterol and Cyclodextrin
[0303] The invention also provides a method for uncoupling from a
membrane an analyte coupled to the membrane using an anchor
comprising cholesterol, comprising contacting the analyte with a
cyclodextrin or a derivative thereof and thereby uncoupling the
analyte from the membrane. Any of the embodiments discussed above,
particularly those concerning the analyte, anchor, cyclodextrin or
a derivative thereof and membrane, are equally applicable to this
method. The analyte is preferably a polynucleotide. The
polynucleotide preferably comprises a leader sequence as defined
above. The cholesterol anchor preferably comprises a polynucleotide
sequence which is hybridised to the leader sequence. The
polynucleotide sequence is preferably covalently attached to the
cholesterol in the anchor.
Kits
[0304] The present invention also provides a kit for determining
the presence, absence or one or more characteristics of two or more
analytes in two or more samples comprising (a) a membrane, (b) one
or more anchors which are capable of coupling the two or more
analytes to the membrane, such as one or more first anchors which
are capable of coupling a first analyte to the membrane and one or
more second anchors which are capable of coupling a second analyte
to the membrane and (c) one or more agents which are capable of
uncoupling at least one of, such as both of, the two or more
analytes from the membrane. The one or more anchors and one or more
agents may be any of those discussed above with reference to the
method of the invention.
[0305] The kit preferably further comprises a detector, such as a
transmembrane pore. Any of the detectors discussed above may be in
the kit.
[0306] The kit preferably further comprises a hairpin loop and/or a
leader sequence which is capable of preferentially threading into a
transmembrane pore. The kit preferably further comprises a
polynucleotide binding protein. Preferred hairpin loops, leader
sequences and polynucleotide binding proteins are discussed
above.
[0307] Any of the embodiments discussed above with reference to the
method of the invention equally apply to the kits. The kit may
further comprise the components of a membrane, such as the
components of an amphiphilic layer or a triblock copolymer
membrane.
[0308] The kit of the invention may additionally comprise one or
more other reagents or instruments which enable any of the
embodiments mentioned above to be carried out. Such reagents or
instruments include one or more of the following: suitable
buffer(s) (aqueous solutions), means to obtain a sample from a
subject (such as a vessel or an instrument comprising a needle),
means to amplify and/or express polynucleotides, a membrane as
defined above or voltage or patch clamp apparatus. Reagents may be
present in the kit in a dry state such that a fluid sample
resuspends the reagents. The kit may also, optionally, comprise
instructions to enable the kit to be used in the method of the
invention or details regarding for which organism the method may be
used.
[0309] The following Examples illustrate the invention.
Examples
Example 1
[0310] This example shows a control experiment which illustrated
that free DNA in solution, which had not been coupled to the
membrane, was not prevented from entering the nanopore by the
presence of methyl-.beta.-cyclodextrin in the experimental
system.
Materials and Methods
[0311] Electrical measurements were acquired from single MspA
nanopores MS(B1-G75S/G77S/L88N/Q126R)8 MspA (MspA-B2C) (SEQ ID NO:
2 with mutations G75S/G77S/L88N/Q126R) inserted in block co-polymer
in buffer (600 mM KCl, 25 mM potassium phosphate, 75 mM potassium
ferrocyanide (II), 25 mM potassium ferricyanide (III) pH 8.0) at a
temperature of 15.degree. C. After achieving a single pore inserted
in the block co-polymer, then buffer (1 mL, 600 mM KCl, 25 mM
potassium phosphate, 75 mM potassium ferrocyanide (II), 25 mM
potassium ferricyanide (III) pH 8.0) was flowed through the system
to remove any excess MspA-B2C nanopores. Two DNA samples (100 nM,
1--SEQ ID NO: 26 and SEQ ID NO: 29 and 2--SEQ ID NO: 27 attached at
its 5' end to four iSp18 spacers which are attached at the opposite
end to the 3' end of SEQ ID NO: 28) were added to the system and
the experiment was run at an applied potential of 120 mV for 30
minutes. The system was then flushed with
methyl-.beta.-cyclodextrin (100 .mu.M) and the DNA samples 1 and 2
at a concentration of 100 nM in a total volume of 500 .mu.L and the
experiment run at an applied potential of 120 mV for a further 30
minutes. The system was then flushed with two 1 mL flushes of
methyl-.beta.-cyclodextrin (100 .mu.M) and the DNA samples 1 and 2
at a concentration of 100 nM.
[0312] A similar control experiment to the one described previously
was carried out except that for all the steps which had contained
methyl-.beta.-cyclodextrin only DNA samples were added and no
methyl-.beta.-cyclodextrin was flushed through the system.
Results
[0313] The control experiment where only free DNA was added to the
system consistently exhibited short spikes in the current trace
which corresponded to the DNA translocating through the nanopore
under the applied potential. This illustrated that DNA
translocation was seen for each flush of the nanopore system with
DNA samples 1 and 2.
[0314] These controls were undertaken to confirm that the reduction
in the number of DNA translocations observed (see Example 4) was
due to the methyl-.beta.-cyclodextrin removing the cholesterol from
the membrane surface, rather than preventing the strand from
entering the pore. The controls tested whether the cyclodextrin
could have bound along the length of the DNA, impeding its ability
to thread through the pore, and thus preventing the strand from
being detected despite the fact that it was still attached to the
membrane. In these experiments, free DNA was used, which had no
anchor to couple it to the membrane, if the interaction of the
cyclodextrin was confined specifically to the cholesterol, the
cyclodextrin should have had no effect on the DNA in this case. Any
reduction in number of DNA translocations observed would,
therefore, have been due to binding of the body of the DNA. No
difference in the number of DNA translocations was observed in the
presence or absence of cyclodextrin, suggesting that the
cyclodextrin present in the system did not bind to the free DNA and
prevent its translocation through the nanopore.
Example 2
[0315] This example shows a further control experiment which
illustrated that when a first sample of coupled DNA was added to
the nanopore system followed by a second sample, without flushing
the system with a de-coupling agent or buffer with no DNA present,
then the number of helicase-controlled DNA movements detected over
a defined period remained fairly constant and helicase controlled
DNA movements were observed for both samples.
Materials and Methods
[0316] The strands used in this study were from a region of the
lambda genome, between 45,042 bp and 48,487 bp. Analytes were made
by the polymerase PCR method to include hybridisation sites at
defined ends of each of the template and template compliment
strands as desired. PCR was carried out from lambda genomic
DNA.
[0317] The DNA template (SEQ ID NO: 31 which corresponds to the
sequence of the strand labelled Alwhich was hybridised to SEQ ID
NO: 47 which corresponds to the sequence for the strand labelled
A2, see FIG. 1(1)) was made using KAPA HiFi 2.times. Master mix,
lambda DNA (NEB) and primers SEQ ID NO: 32 and SEQ ID NO: 33.
Reactions were cycled 20 times and product of the correct size was
purified by Gel Filtration on Sephacryl S1000 column and
concentrated to 0.25 mg/ml using Millipore Ultracel 15 50 kDa
concentrators.
[0318] DNA constructs (X and Y) for electrophysiology experiments
were made according to the same reaction mix; 2.times. LongAmp Taq
master mix, 300 nM of primers 1 and 2 or 3 and 4, 1.2 ng ul.sup.-1
DNA template (SEQ ID NO: 31 which corresponds to the sequence for
the strand labelled Alwhich was hybridised to SEQ ID NO: 47 which
corresponds to the sequence for the strand labelled A2, see FIG.
1(1)). DNA constructs were all amplified according to the same
cycling program; 94.degree. C. for 2 mins, [94.degree. C. for 15
secs, 58.degree. C. for 30 secs, 65.degree. C. for 2 mins].sub.12
and 65.degree. C. for 5 mins. DNA constructs were all purified from
a 0.8% agarose gel according to manufacturer's instructions (Qiagen
Gel Extraction kit) and then SPRI purified (Agencourt AMPure beads)
according to manufacturer's instructions.
[0319] For DNA construct X=SEQ ID NO: 34 attached at its 3' end to
four iSpC3 spacers which are attached at the opposite end to the 5'
end of SEQ ID NO: 35; SEQ ID NO: 35 is attached at the 3' end to
four iSpC3 spacers which are attached at the opposite end to four
5-nitroindoles which are attached to the 3' end of SEQ ID NO: 39.
The primers used to produce construct X are primer 1--SEQ ID NO: 34
attached at the 3' end to four iSpC3 spacers which are attached at
the opposite end to the 5' end of SEQ ID NO: 35; SEQ ID NO: 35 is
also attached at the 3' end to four iSpC3 spacers which are
attached at the opposite end to four 5-nitroindoles which are
attached to the 5' end of SEQ ID NO: 36 and primer 2--SEQ ID NO: 37
is attached at the 3' end to four 5-nitroindoles which are attached
at the opposite end to the 5' end of SEQ ID NO: 38.
[0320] Construct X was then hybridised to SEQ ID NO: 41 and SEQ ID
NO: 41 which is attached at the 3' end to six iSp18 spacers which
are attached at the opposite end to two thymines and a 3'
cholesterol TEG (FIG. 1(2) shows a cartoon representation of
construct X). The tethers were annealed at a five-fold excess at
room temperature for ten minutes in 25 mM potassium phosphate
buffer, 151 mM potassium chloride, pH 8.0.
[0321] For DNA construct Y=SEQ ID NO: 34 attached at its 3' end to
four iSpC3 spacers which are attached at the opposite end to the 5'
end of SEQ ID NO: 37; SEQ ID NO: 37 is attached at the 3' end to
four iSpC3 spacers which are attached at the opposite end to four
5-nitroindoles which are attached to the 5' end of SEQ ID NO: 40.
The primers used to produce construct Y are primer 3--SEQ ID NO: 34
attached at the 3' end to four iSpC3 spacers which are attached at
the opposite end to the 5' end of SEQ ID NO: 37; SEQ ID NO: 37 is
attached at the 3' end to four iSpC3 spacers which are attached at
the opposite end to four 5-nitroindoles which are attached to the
5' end of SEQ ID NO: 38 and primer 4--SEQ ID NO: 35 is attached at
the 3' end to four 5-nitroindoles which are attached at the
opposite end to the 5' end of SEQ ID NO: 36.
[0322] Construct Y was then hybridised to SEQ ID NO: 30 and SEQ ID
NO: 30 which is attached at the 3' end to six iSp18 spacers which
are attached at the opposite end to two thymines and a 3'
cholesterol TEG (FIG. 1(3) shows a cartoon representation of
construct Y). The tethers were annealed at a five-fold excess at
room temperature for ten minutes in 25 mM potassium phosphate
buffer, 151 mM potassium chloride, pH 8.0.
[0323] Prior to setting up the experiment, the DNA constructs X and
Y with their appropriate tethers (stock concentration 20 nM, final
concentration added to nanopore system 0.1 nM) were separately
incubated with reagents as described. Firstly the DNA was
pre-incubated at room temperature for five minutes with T4
Dda-E94C/A360C (stock concentration 250 nM, final concentration
added to nanopore system 1 nM, SEQ ID NO: 24 with mutations
E94C/A360C) in buffer (151 mM KCl, 25 mM phosphate, 2 mM EDTA,
pH8.0). After five minutes TMAD (500 .mu.M) was added to the
pre-mix and the mixture incubated for a further 5 minutes. Finally,
MgCl2 (10 mM final concentration), ATP (2.5 mM final concentration)
and buffer (150 mM potassium ferrocyanide (II), 150 mM potassium
ferricyanide and 25 mM potassium phosphate pH 8.0) were added to
the pre-mix.
[0324] Electrical measurements were acquired from single MspA
nanopores (MspA-B2C) inserted in block co-polymer in buffer (25 mM
potassium phosphate, 150 mM potassium ferrocyanide (II), 150 mM
potassium ferricyanide (III), pH 8.0). After achieving a single
pore inserted in the block co-polymer, then buffer (2 mL, 25 mM
potassium phosphate pH 8.0, 150 mM potassium ferrocyanide (II) and
150 mM potassium ferricyanide (III)) was flowed through the system
to remove any excess MspA nanopores. The enzyme (T4 Dda-E94C/A360C,
1 nM final concentration), DNA construct X (0.1 nM final
concentration), fuel (MgCl2 10 mM final concentration, ATP 2.5 mM
final concentration) pre-mix (150 .mu.L total) was then added to
the single nanopore experimental system and the experiment was run
at a holding potential of 120 mV for 2 hours and
helicase-controlled DNA movement was monitored. After two hours,
the experimental protocol was stopped, the potential set to zero
and the DNA construct Y/enzyme pre-mix (150 .mu.L total) was then
added directly to the system with no de-coupling agents or flushes
of buffer included. The experiment was then run for a further 2
hours at a holding potential of 120 mV and helicase controlled DNA
movement monitored.
Results and Discussion
[0325] DNA contructs X and Y (shown in FIGS. 1 (a) and (b)
respectively) were prepared from the same 3.8 kB section of the
lambda phage genome. Adaptors were attached to give an overhanging
"leader" at one end of the duplex, which allowed capture and
threading by the pore as well as providing an enzyme binding site.
The other end was left blunt so only the strand with the leader on
was captured and sequenced. The two samples had the adaptor ligated
to opposite ends, such that the leader was joined to strand A1
(shown in FIG. 1(2)) in DNA construct X and to strand A2 (shown in
FIG. 1(3)) in DNA construct Y. This meant that DNA constructs X and
Y gave detectable strand movements with sequences that mapped only
to distinct regions of the lambda genome. These movements were
easily distinguished, so provided a convenient way of identifying
two different test samples; however any other samples with
different sequences could have been used just as well.
[0326] Helicase controlled DNA movement was observed for both DNA
constructs X and Y, with T4 Dda-E94C/A360C. Figure two shows the
experimental time course with the percentage of time the nanopores
were present in their unblocked state (shown as light grey)
compared to when a helicase DNA movement was occurring and the
nanopores were partially blocked by the DNA strand (shown as
black). For the first 2100 seconds no DNA was present in the
system, therefore, the nanopores were in an unblocked state. DNA
construct X was added at 2400 seconds and helicase controlled DNA
movements were occurring through the nanopore around 80% of the
time. DNA construct Y was then flowed into the nanopore system at
7200 seconds and again helicase controlled DNA movement through the
nanopore was observed for approximately 80% of the time.
[0327] Upon the addition of construct X, the helicase controlled
DNA movements observed were all identified as corresponding to this
construct. When construct Y was flowed into the system helicase
controlled DNA movements corresponding to Y were detected as well
as a significant number of movements which corresponded to
construct X. Experimental data showed that the rate of helicase
controlled DNA movements detected remained fairly constant
throughout the experiment and that by adding construct Y to the
system, with no additional flushing or de-coupling agents, helicase
controlled DNA movements were detected for both samples.
Example 3
[0328] This example illustrates that when coupled DNA construct X
was added to the nanopore system it was not possible to remove the
sample simply by flushing the system with a large volume of
buffer.
Materials and Methods
[0329] DNA constructs X and Y were prepared as described in Example
2. The DNA constructs were pre-incubated with enzyme as described
in Example 2 producing the construct X and construct Y
pre-mixes.
[0330] The nanopore experimental system was set up as described in
Example 2. DNA construct X/enzyme pre-mix (300 .mu.L total) was
added to the experimental system and the experiment run at a
holding potential of 120 mV for two hours and helicase controlled
DNA movement was monitored. After two hours, the experimental
protocol was stopped, the potential set to zero and buffer (10 mL
of 150 mM potassium ferrocyanide (II), 150 mM potassium
ferricyanide (III), 25 mM potassium phosphate pH 8.0) was flowed
through the nanopore system in order to try and remove coupled DNA
construct X. After the buffer flush, the experiment was run with no
additional DNA added to the system at a holding potential of 120 mV
for two hours and helicase controlled DNA movement was monitored.
Finally, DNA construct Y/enzyme pre-mix (300 .mu.L total) was added
to the experimental system and the experiment run at a holding
potential of 120 mV for two hours and helicase controlled DNA
movement was monitored.
Results and Discussion
[0331] Helicase controlled DNA movement was observed for both DNA
constructs X and Y, with T4 Dda-E94C/A360C. FIG. 3 shows part of
the experimental time course with the percentage of time the
nanopores were present in their unblocked state (shown as light
grey) compared to when a helicase DNA movement was occurring and
the nanopores were partially blocked by the DNA strand (shown as
black). For the first 2400 seconds no DNA was present in the
system, therefore, the nanopores were in an unblocked state. DNA
construct X was added at 2700 seconds and helicase controlled DNA
movements were occurring through the nanopore around 80% of the
time. Buffer (10 mL) was flowed across the system, at 7500 seconds
and then the percentage of time the nanopore was partially blocked
owing to helicase controlled DNA movement was then monitored. After
flushing with buffer, helicase controlled DNA movements were
occurring through the nanopore around 50% of the time. This
indicated that the amount of coupled DNA construct X present in the
system had been reduced by the buffer flush, however, a large
number of helicase controlled DNA movements were still detected.
Upon the addition of DNA construct Y, helicase controlled DNA
movements corresponding to Y were detected as well as a significant
number of helicase controlled DNA movements which corresponded to
construct X which was still present in the system.
Example 4
[0332] This example illustrates how methyl-.beta.-cyclodextrin was
used to decouple DNA, which was coupled to the membrane using a
cholesterol TEG, from the membrane. A solution of
methyl-.beta.-cyclodextrin was added to the nanopore system for 1,
10 and 30 mins and the number of helicase-controlled DNA movements
detected over a defined period was monitored after each incubation.
This experiment illustrated that even using an incubation period of
only one minute significant decoupling of the DNA from the membrane
was detected.
Materials and Methods
[0333] DNA constructs X and Y were prepared as described in Example
2. The DNA constructs were pre-incubated with enzyme as described
in Example 2 producing the construct X and construct Y
pre-mixes.
[0334] The nanopore experimental system was set up as described in
Example 2. DNA construct X/enzyme pre-mix (150 .mu.L total) was
added to the experimental system and the experiment run at a
holding potential of 120 mV for two hours and helicase controlled
DNA movement was monitored. After two hours, the experimental
protocol was stopped, the potential set to zero and
methyl-.beta.-cyclodextrin (150 .mu.L of 100 .mu.M) was flowed onto
the nanopore system and incubated for 1, 10 or 30 minutes in order
to try and remove coupled DNA construct X. After the appropriate
incubation period, buffer (150 .mu.L, 150 mM potassium ferrocyanide
(II), 150 mM potassium ferricyanide (III), 25 mM potassium
phosphate, pH 8.0) was flushed through the system to remove any
de-coupled DNA and excess methyl-.beta.-cyclodextrin. After the
buffer flush, the experiment was run with no additional DNA added
to the system at a holding potential of 120 mV for two hours and
helicase controlled DNA movement was monitored. Finally, DNA
construct Y/enzyme pre-mix (150 .mu.L total) was added to the
experimental system and the experiment run at a holding potential
of 120 mV for two hours and helicase controlled DNA movement was
monitored. The same de-coupling procedure was then repeated for DNA
construct Y.
Results and Discussion
[0335] Helicase controlled DNA movement was observed for both DNA
constructs X and Y, with T4 Dda-E94C/A360C. FIGS. 4, 5 and 6 show
part of the experimental time course with the percentage of time
the nanopores were present in their unblocked state (shown as light
grey) compared to when a helicase DNA movement was occurring and
the nanopores were partially blocked by the DNA strand (shown as
black). FIGS. 4, 5 and 6 correspond to incubation periods of 1, 10
and 30 minutes with methyl-.beta.-cyclodextrin respectively. For
all three experiments, prior to addition of DNA, little or no
helicase controlled DNA movements were observed. Upon the addition
of construct X helicase controlled DNA movements were occurring
through the nanopore around 80% of the time. After the addition of
methyl-.beta.-cyclodextrin for various incubation periods and the
corresponding buffer flush, the percentage of time the nanopore was
partially blocked owing to helicase controlled DNA movement was
drastically reduced to around 20% and for the 30 minute incubation
to less than 10%. This indicated that methyl-.beta.-cyclodextrin
successfully decoupled DNA, which had been coupled to a membrane
using cholesterol, from the membrane. The
methyl-.beta.-cyclodextrin decoupled significantly more coupled DNA
than flushing with buffer.
[0336] Upon the addition of DNA construct Y to the system, helicase
controlled DNA movements which corresponded to construct Y were
identified. A small proportion of movements were identified as
corresponding to construct X, however, the proportion of events
identified as X was significantly reduced in comparison to
experiments where either construct X was not flushed from the
system (see Example 2) or where tethered construct X was treated
with 10 mL of buffer in an attempt to remove it from the system
(see Example 3). The methyl-.beta.-cyclodextrin decoupling process
was repeated for construct Y and it was also shown that it was
possible to successfully decouple construct Y from the membrane
using this method.
Example 5
[0337] This example illustrates how DNA, which has had a
biotin-tether hybridised onto it and has been pre-incubated with
streptavidin, has been coupled to the membrane by the streptavidin
binding a 5' desthiobiotin of an extender (which also has a
cholesterol at the 3' end) (see FIG. 7 for cartoon representation).
This DNA construct can then be decoupled from the membrane by
flushing the system, with free biotin. As biotin has a stronger
binding affinity for streptavidin than desthiobiotin when the
biotin was added to the system it out competed the desthiobiotin,
which ensured efficient removal of the strand. This left the
extenders coupled to the bilayer and available for coupling of a
second DNA construct to the membrane.
Materials and Methods
[0338] The DNA construct used in Example 5 is shown in FIG. 7. The
DNA construct was prepared by hybridising SEQ ID NO: 45 (50 nM,
which has six iSp18 spacers attached to its 3' end which are
attached at the opposite end to two thymines and a 3'biotin TEG) to
the DNA strand which was made up of SEQ ID NO: 42 which is attached
at its 3' end to four iSpC3 spacers which are attached at the
opposite end to the 5' end of SEQ ID NO: 43 (50 nM) at 50.degree.
C. for ten minutes and then slow cooled. Streptavidin (final
concentration 50 nM) was added to the DNA mixture (final
concentration 25 nM) and incubated at room temperature for 10
minutes. This complex will be referred to as DNA construct P.
[0339] The nanopore experimental system was set up as described in
Example 2. A control experiment was run, with no DNA added to the
system for 15 minutes at an applied potential of 120 mV. The
desthiobiotin extender (SEQ ID NO: 46 which has a desthiobiotin
attached at the 5' end and a cholesterol TEG attached at the 3'
end) was then added to the nanopore system and the experiment run
for 15 mins allowing it to couple to the membrane. DNA construct P
was added to the experimental system (25 nM) and the experiment run
at an applied potential of 120 mV for 30 minutes. Free biotin (50
.mu.M) was then added to the system and the experiment run for a
further 30 minutes. After the biotin incubation, buffer was flowed
through the system (1 mL, 625 mM KCl, 100 mM HEPES, pH8) to remove
any excess biotin and de-coupled DNA.
Results and Discussion
[0340] This experiment illustrates another method for de-coupling
DNA constructs from a membrane. FIG. 8 shows the current trace of
the full experiment described above. FIG. 9 shows several
continuous snap shots of the experimental steps described
previously. FIG. 9(A) initially shows that the nanopore was open
and exhibited a couple of blocks when no DNA was present in the
system. *1 marks the point in the experiment when the desthiobiotin
extended was added to the system, current blocks corresponding to
this short fragment were not observed. *2 marks the point where DNA
construct P was added to the system. The addition of DNA resulted
in DNA current blocks which were consistently between 70 and 100 pA
(see FIG. 9A (last portion of the trace and 9B the first portion of
the trace). *3 marks the point where biotin (50 .mu.M) was added to
the system. It was clear that upon addition of biotin there was a
drastic reduction in the number of DNA current blocks observed.
Finally, *4 corresponds to the buffer flush step where the DNA and
biotin were removed from the system. This experiment illustrated
that by flushing biotin into the system DNA construct P could be
de-coupled from the membrane. As biotin has a stronger binding
affinity for streptavidin than desthiobiotin when the biotin was
added to the system it out competed the desthiobiotin, which
ensured efficient removal of the strand. The biotin also bound to
the other free binding sites on the streptavidin and the
whole-streptavidin DNA complex was removed from the system. This
left the extenders coupled to the bilayer and available for
coupling of a second DNA construct to the membrane.
Example 6
[0341] This example illustrates how
(2-hydroxypropyl)-.beta.-cyclodextrin was used to decouple DNA,
which was coupled to the membrane using a cholesterol TEG, from the
membrane. Various different concentrations of
(2-hydroxypropyl)-.beta.-cyclodextrin were added to the nanopore
system and the % change in the number of helicase controlled DNA
movements that were detected per nanopore over a defined period was
monitored. This experiment illustrated that concentrations as low
as 20 mM (2-hydroxypropyl)-.beta.-cyclodextrin resulted in a
reduction in the number of helicase controlled DNA movements
detected per nanopore, after incubation (see Table 2).
Materials and Methods
[0342] DNA construct X was prepared as described in Example 2. The
DNA construct was pre-incubated with enzyme as described in Example
2 producing the construct X pre-mix.
[0343] The nanopore experimental system was set up as described in
Example 2. DNA construct X/enzyme pre-mix (150 .mu.L total) was
added to the experimental system and the experiment run at a
holding potential of 140 mV for two hours and helicase controlled
DNA movement was monitored. After two hours, the experimental
protocol was stopped, the potential set to zero and
(2-hydroxypropyl)-.beta.-cyclodextrin (150 .mu.L of either 20 mM,
50 mM, 100 mM or 200 mM in 500 mM KCl, 25 mM K Phosphate pH8) was
flowed onto the nanopore system and incubated for 10 minutes in
order to try to remove coupled DNA construct X. After the
incubation period, buffer (150 .mu.L, 500 mM KCl, 25 mM K Phosphate
pH8) was flushed through the system to remove any de-coupled DNA
and excess (2-hydroxypropyl)-.beta.-cyclodextrin. After the buffer
flush, buffer (containing fuel) was added to the system (150 uL of
500 mM KCl, 25 mM K Phosphate 2 mM ATP, 2 mM MgCl2 pH8) with no
additional DNA at a holding potential of 140 mV for two hours and
helicase controlled DNA movement was monitored.
Results and Discussion
[0344] Helicase controlled DNA movement was observed for DNA
construct X, with T4 Dda-E94C/A360C. Table 2 below shows the
average % change in the number of helicase controlled DNA movements
that were detected per nanopore, after the system had been
incubated with (2-hydroxypropyl)-.beta.-cyclodextrin at various
concentrations. For all experiments, prior to addition of DNA, few
or no helicase controlled DNA movements were observed. Upon the
addition of construct X, helicase controlled DNA movements were
occurring through the nanopore. After the addition of
(2-hydroxypropyl)-.beta.-cyclodextrin at various concentrations and
the corresponding buffer flush, the average percentage change in
the number of helicase controlled DNA movements that were detected
per nanopore was at least 50% and was as much as 90% when incubated
at 200 mM concentration. This indicated that
(2-hydroxypropyl)-.beta.-cyclodextrin successfully decoupled DNA,
which had been coupled to a membrane using cholesterol, from the
membrane.
TABLE-US-00002 TABLE 2 Reduction in the number of helicase
controlled DNA movements detected (Average % change per nanopore)
Concentration Experiment 1 Experiment 2 20 mM -53.73 -52.39 50 mM
-59.23 -79.10 100 mM -84.06 -83.38 200 mM -90.90 -96.20
Sequence CWU 1
1
471558DNAArtificial SequenceMycobacterium smegmatis porin A mutant
(D90N/D91N/D93N/D118R/D134R/E193K) 1atgggtctgg ataatgaact
gagcctggtg gacggtcaag atcgtaccct gacggtgcaa 60caatgggata cctttctgaa
tggcgttttt ccgctggatc gtaatcgcct gacccgtgaa 120tggtttcatt
ccggtcgcgc aaaatatatc gtcgcaggcc cgggtgctga cgaattcgaa
180ggcacgctgg aactgggtta tcagattggc tttccgtggt cactgggcgt
tggtatcaac 240ttctcgtaca ccacgccgaa tattctgatc aacaatggta
acattaccgc accgccgttt 300ggcctgaaca gcgtgattac gccgaacctg
tttccgggtg ttagcatctc tgcccgtctg 360ggcaatggtc cgggcattca
agaagtggca acctttagtg tgcgcgtttc cggcgctaaa 420ggcggtgtcg
cggtgtctaa cgcccacggt accgttacgg gcgcggccgg cggtgtcctg
480ctgcgtccgt tcgcgcgcct gattgcctct accggcgaca gcgttacgac
ctatggcgaa 540ccgtggaata tgaactaa 5582184PRTArtificial
SequenceMycobacterium smegmatis porin A mutant
(D90N/D91N/D93N/D118R/D134R/E139K) 2Gly Leu Asp Asn Glu Leu Ser Leu
Val Asp Gly Gln Asp Arg Thr Leu 1 5 10 15 Thr Val Gln Gln Trp Asp
Thr Phe Leu Asn Gly Val Phe Pro Leu Asp 20 25 30 Arg Asn Arg Leu
Thr Arg Glu Trp Phe His Ser Gly Arg Ala Lys Tyr 35 40 45 Ile Val
Ala Gly Pro Gly Ala Asp Glu Phe Glu Gly Thr Leu Glu Leu 50 55 60
Gly Tyr Gln Ile Gly Phe Pro Trp Ser Leu Gly Val Gly Ile Asn Phe 65
70 75 80 Ser Tyr Thr Thr Pro Asn Ile Leu Ile Asn Asn Gly Asn Ile
Thr Ala 85 90 95 Pro Pro Phe Gly Leu Asn Ser Val Ile Thr Pro Asn
Leu Phe Pro Gly 100 105 110 Val Ser Ile Ser Ala Arg Leu Gly Asn Gly
Pro Gly Ile Gln Glu Val 115 120 125 Ala Thr Phe Ser Val Arg Val Ser
Gly Ala Lys Gly Gly Val Ala Val 130 135 140 Ser Asn Ala His Gly Thr
Val Thr Gly Ala Ala Gly Gly Val Leu Leu 145 150 155 160 Arg Pro Phe
Ala Arg Leu Ile Ala Ser Thr Gly Asp Ser Val Thr Thr 165 170 175 Tyr
Gly Glu Pro Trp Asn Met Asn 180 3885DNAArtificial
Sequencealpha-hemolysin mutant (E111N/K147N) 3atggcagatt ctgatattaa
tattaaaacc ggtactacag atattggaag caatactaca 60gtaaaaacag gtgatttagt
cacttatgat aaagaaaatg gcatgcacaa aaaagtattt 120tatagtttta
tcgatgataa aaatcacaat aaaaaactgc tagttattag aacaaaaggt
180accattgctg gtcaatatag agtttatagc gaagaaggtg ctaacaaaag
tggtttagcc 240tggccttcag cctttaaggt acagttgcaa ctacctgata
atgaagtagc tcaaatatct 300gattactatc caagaaattc gattgataca
aaaaactata tgagtacttt aacttatgga 360ttcaacggta atgttactgg
tgatgataca ggaaaaattg gcggccttat tggtgcaaat 420gtttcgattg
gtcatacact gaactatgtt caacctgatt tcaaaacaat tttagagagc
480ccaactgata aaaaagtagg ctggaaagtg atatttaaca atatggtgaa
tcaaaattgg 540ggaccatacg atcgagattc ttggaacccg gtatatggca
atcaactttt catgaaaact 600agaaatggtt ctatgaaagc agcagataac
ttccttgatc ctaacaaagc aagttctcta 660ttatcttcag ggttttcacc
agacttcgct acagttatta ctatggatag aaaagcatcc 720aaacaacaaa
caaatataga tgtaatatac gaacgagttc gtgatgatta ccaattgcat
780tggacttcaa caaattggaa aggtaccaat actaaagata aatggacaga
tcgttcttca 840gaaagatata aaatcgattg ggaaaaagaa gaaatgacaa attaa
8854293PRTArtificial Sequencealpha-hemolysin mutant (E111N/K147N)
4Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly Ser 1
5 10 15 Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu
Asn 20 25 30 Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp
Lys Asn His 35 40 45 Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly
Thr Ile Ala Gly Gln 50 55 60 Tyr Arg Val Tyr Ser Glu Glu Gly Ala
Asn Lys Ser Gly Leu Ala Trp 65 70 75 80 Pro Ser Ala Phe Lys Val Gln
Leu Gln Leu Pro Asp Asn Glu Val Ala 85 90 95 Gln Ile Ser Asp Tyr
Tyr Pro Arg Asn Ser Ile Asp Thr Lys Asn Tyr 100 105 110 Met Ser Thr
Leu Thr Tyr Gly Phe Asn Gly Asn Val Thr Gly Asp Asp 115 120 125 Thr
Gly Lys Ile Gly Gly Leu Ile Gly Ala Asn Val Ser Ile Gly His 130 135
140 Thr Leu Asn Tyr Val Gln Pro Asp Phe Lys Thr Ile Leu Glu Ser Pro
145 150 155 160 Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe Asn Asn
Met Val Asn 165 170 175 Gln Asn Trp Gly Pro Tyr Asp Arg Asp Ser Trp
Asn Pro Val Tyr Gly 180 185 190 Asn Gln Leu Phe Met Lys Thr Arg Asn
Gly Ser Met Lys Ala Ala Asp 195 200 205 Asn Phe Leu Asp Pro Asn Lys
Ala Ser Ser Leu Leu Ser Ser Gly Phe 210 215 220 Ser Pro Asp Phe Ala
Thr Val Ile Thr Met Asp Arg Lys Ala Ser Lys 225 230 235 240 Gln Gln
Thr Asn Ile Asp Val Ile Tyr Glu Arg Val Arg Asp Asp Tyr 245 250 255
Gln Leu His Trp Thr Ser Thr Asn Trp Lys Gly Thr Asn Thr Lys Asp 260
265 270 Lys Trp Thr Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp Trp Glu
Lys 275 280 285 Glu Glu Met Thr Asn 290 5184PRTMycobacterium
smegmatis 5Gly Leu Asp Asn Glu Leu Ser Leu Val Asp Gly Gln Asp Arg
Thr Leu 1 5 10 15 Thr Val Gln Gln Trp Asp Thr Phe Leu Asn Gly Val
Phe Pro Leu Asp 20 25 30 Arg Asn Arg Leu Thr Arg Glu Trp Phe His
Ser Gly Arg Ala Lys Tyr 35 40 45 Ile Val Ala Gly Pro Gly Ala Asp
Glu Phe Glu Gly Thr Leu Glu Leu 50 55 60 Gly Tyr Gln Ile Gly Phe
Pro Trp Ser Leu Gly Val Gly Ile Asn Phe 65 70 75 80 Ser Tyr Thr Thr
Pro Asn Ile Leu Ile Asp Asp Gly Asp Ile Thr Ala 85 90 95 Pro Pro
Phe Gly Leu Asn Ser Val Ile Thr Pro Asn Leu Phe Pro Gly 100 105 110
Val Ser Ile Ser Ala Asp Leu Gly Asn Gly Pro Gly Ile Gln Glu Val 115
120 125 Ala Thr Phe Ser Val Asp Val Ser Gly Pro Ala Gly Gly Val Ala
Val 130 135 140 Ser Asn Ala His Gly Thr Val Thr Gly Ala Ala Gly Gly
Val Leu Leu 145 150 155 160 Arg Pro Phe Ala Arg Leu Ile Ala Ser Thr
Gly Asp Ser Val Thr Thr 165 170 175 Tyr Gly Glu Pro Trp Asn Met Asn
180 6184PRTMycobacterium smegmatis 6Gly Leu Asp Asn Glu Leu Ser Leu
Val Asp Gly Gln Asp Arg Thr Leu 1 5 10 15 Thr Val Gln Gln Trp Asp
Thr Phe Leu Asn Gly Val Phe Pro Leu Asp 20 25 30 Arg Asn Arg Leu
Thr Arg Glu Trp Phe His Ser Gly Arg Ala Lys Tyr 35 40 45 Ile Val
Ala Gly Pro Gly Ala Asp Glu Phe Glu Gly Thr Leu Glu Leu 50 55 60
Gly Tyr Gln Ile Gly Phe Pro Trp Ser Leu Gly Val Gly Ile Asn Phe 65
70 75 80 Ser Tyr Thr Thr Pro Asn Ile Leu Ile Asp Asp Gly Asp Ile
Thr Gly 85 90 95 Pro Pro Phe Gly Leu Glu Ser Val Ile Thr Pro Asn
Leu Phe Pro Gly 100 105 110 Val Ser Ile Ser Ala Asp Leu Gly Asn Gly
Pro Gly Ile Gln Glu Val 115 120 125 Ala Thr Phe Ser Val Asp Val Ser
Gly Pro Ala Gly Gly Val Ala Val 130 135 140 Ser Asn Ala His Gly Thr
Val Thr Gly Ala Ala Gly Gly Val Leu Leu 145 150 155 160 Arg Pro Phe
Ala Arg Leu Ile Ala Ser Thr Gly Asp Ser Val Thr Thr 165 170 175 Tyr
Gly Glu Pro Trp Asn Met Asn 180 7183PRTMycobacterium smegmatis 7Val
Asp Asn Gln Leu Ser Val Val Asp Gly Gln Gly Arg Thr Leu Thr 1 5 10
15 Val Gln Gln Ala Glu Thr Phe Leu Asn Gly Val Phe Pro Leu Asp Arg
20 25 30 Asn Arg Leu Thr Arg Glu Trp Phe His Ser Gly Arg Ala Thr
Tyr His 35 40 45 Val Ala Gly Pro Gly Ala Asp Glu Phe Glu Gly Thr
Leu Glu Leu Gly 50 55 60 Tyr Gln Val Gly Phe Pro Trp Ser Leu Gly
Val Gly Ile Asn Phe Ser 65 70 75 80 Tyr Thr Thr Pro Asn Ile Leu Ile
Asp Gly Gly Asp Ile Thr Gln Pro 85 90 95 Pro Phe Gly Leu Asp Thr
Ile Ile Thr Pro Asn Leu Phe Pro Gly Val 100 105 110 Ser Ile Ser Ala
Asp Leu Gly Asn Gly Pro Gly Ile Gln Glu Val Ala 115 120 125 Thr Phe
Ser Val Asp Val Lys Gly Ala Lys Gly Ala Val Ala Val Ser 130 135 140
Asn Ala His Gly Thr Val Thr Gly Ala Ala Gly Gly Val Leu Leu Arg 145
150 155 160 Pro Phe Ala Arg Leu Ile Ala Ser Thr Gly Asp Ser Val Thr
Thr Tyr 165 170 175 Gly Glu Pro Trp Asn Met Asn 180
81830DNABacillus subtilis phage phi29 8atgaaacaca tgccgcgtaa
aatgtatagc tgcgcgtttg aaaccacgac caaagtggaa 60gattgtcgcg tttgggccta
tggctacatg aacatcgaag atcattctga atacaaaatc 120ggtaacagtc
tggatgaatt tatggcatgg gtgctgaaag ttcaggcgga tctgtacttc
180cacaacctga aatttgatgg cgcattcatt atcaactggc tggaacgtaa
tggctttaaa 240tggagcgcgg atggtctgcc gaacacgtat aataccatta
tctctcgtat gggccagtgg 300tatatgattg atatctgcct gggctacaaa
ggtaaacgca aaattcatac cgtgatctat 360gatagcctga aaaaactgcc
gtttccggtg aagaaaattg cgaaagattt caaactgacg 420gttctgaaag
gcgatattga ttatcacaaa gaacgtccgg ttggttacaa aatcaccccg
480gaagaatacg catacatcaa aaacgatatc cagatcatcg cagaagcgct
gctgattcag 540tttaaacagg gcctggatcg catgaccgcg ggcagtgata
gcctgaaagg tttcaaagat 600atcatcacga ccaaaaaatt caaaaaagtg
ttcccgacgc tgagcctggg tctggataaa 660gaagttcgtt atgcctaccg
cggcggtttt acctggctga acgatcgttt caaagaaaaa 720gaaattggcg
agggtatggt gtttgatgtt aatagtctgt atccggcaca gatgtacagc
780cgcctgctgc cgtatggcga accgatcgtg ttcgagggta aatatgtttg
ggatgaagat 840tacccgctgc atattcagca catccgttgt gaatttgaac
tgaaagaagg ctatattccg 900accattcaga tcaaacgtag tcgcttctat
aagggtaacg aatacctgaa aagctctggc 960ggtgaaatcg cggatctgtg
gctgagtaac gtggatctgg aactgatgaa agaacactac 1020gatctgtaca
acgttgaata catcagcggc ctgaaattta aagccacgac cggtctgttc
1080aaagatttca tcgataaatg gacctacatc aaaacgacct ctgaaggcgc
gattaaacag 1140ctggccaaac tgatgctgaa cagcctgtat ggcaaattcg
cctctaatcc ggatgtgacc 1200ggtaaagttc cgtacctgaa agaaaatggc
gcactgggtt ttcgcctggg cgaagaagaa 1260acgaaagatc cggtgtatac
cccgatgggt gttttcatta cggcctgggc acgttacacg 1320accatcaccg
cggcccaggc atgctatgat cgcattatct actgtgatac cgattctatt
1380catctgacgg gcaccgaaat cccggatgtg attaaagata tcgttgatcc
gaaaaaactg 1440ggttattggg cccacgaaag tacgtttaaa cgtgcaaaat
acctgcgcca gaaaacctac 1500atccaggata tctacatgaa agaagtggat
ggcaaactgg ttgaaggttc tccggatgat 1560tacaccgata tcaaattcag
tgtgaaatgc gccggcatga cggataaaat caaaaaagaa 1620gtgaccttcg
aaaacttcaa agttggtttc agccgcaaaa tgaaaccgaa accggtgcag
1680gttccgggcg gtgtggttct ggtggatgat acgtttacca ttaaatctgg
cggtagtgcg 1740tggagccatc cgcagttcga aaaaggcggt ggctctggtg
gcggttctgg cggtagtgcc 1800tggagccacc cgcagtttga aaaataataa
18309608PRTBacillus subtilis phage phi29 9Met Lys His Met Pro Arg
Lys Met Tyr Ser Cys Ala Phe Glu Thr Thr 1 5 10 15 Thr Lys Val Glu
Asp Cys Arg Val Trp Ala Tyr Gly Tyr Met Asn Ile 20 25 30 Glu Asp
His Ser Glu Tyr Lys Ile Gly Asn Ser Leu Asp Glu Phe Met 35 40 45
Ala Trp Val Leu Lys Val Gln Ala Asp Leu Tyr Phe His Asn Leu Lys 50
55 60 Phe Asp Gly Ala Phe Ile Ile Asn Trp Leu Glu Arg Asn Gly Phe
Lys 65 70 75 80 Trp Ser Ala Asp Gly Leu Pro Asn Thr Tyr Asn Thr Ile
Ile Ser Arg 85 90 95 Met Gly Gln Trp Tyr Met Ile Asp Ile Cys Leu
Gly Tyr Lys Gly Lys 100 105 110 Arg Lys Ile His Thr Val Ile Tyr Asp
Ser Leu Lys Lys Leu Pro Phe 115 120 125 Pro Val Lys Lys Ile Ala Lys
Asp Phe Lys Leu Thr Val Leu Lys Gly 130 135 140 Asp Ile Asp Tyr His
Lys Glu Arg Pro Val Gly Tyr Lys Ile Thr Pro 145 150 155 160 Glu Glu
Tyr Ala Tyr Ile Lys Asn Asp Ile Gln Ile Ile Ala Glu Ala 165 170 175
Leu Leu Ile Gln Phe Lys Gln Gly Leu Asp Arg Met Thr Ala Gly Ser 180
185 190 Asp Ser Leu Lys Gly Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe
Lys 195 200 205 Lys Val Phe Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu
Val Arg Tyr 210 215 220 Ala Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp
Arg Phe Lys Glu Lys 225 230 235 240 Glu Ile Gly Glu Gly Met Val Phe
Asp Val Asn Ser Leu Tyr Pro Ala 245 250 255 Gln Met Tyr Ser Arg Leu
Leu Pro Tyr Gly Glu Pro Ile Val Phe Glu 260 265 270 Gly Lys Tyr Val
Trp Asp Glu Asp Tyr Pro Leu His Ile Gln His Ile 275 280 285 Arg Cys
Glu Phe Glu Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln Ile 290 295 300
Lys Arg Ser Arg Phe Tyr Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly 305
310 315 320 Gly Glu Ile Ala Asp Leu Trp Leu Ser Asn Val Asp Leu Glu
Leu Met 325 330 335 Lys Glu His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile
Ser Gly Leu Lys 340 345 350 Phe Lys Ala Thr Thr Gly Leu Phe Lys Asp
Phe Ile Asp Lys Trp Thr 355 360 365 Tyr Ile Lys Thr Thr Ser Glu Gly
Ala Ile Lys Gln Leu Ala Lys Leu 370 375 380 Met Leu Asn Ser Leu Tyr
Gly Lys Phe Ala Ser Asn Pro Asp Val Thr 385 390 395 400 Gly Lys Val
Pro Tyr Leu Lys Glu Asn Gly Ala Leu Gly Phe Arg Leu 405 410 415 Gly
Glu Glu Glu Thr Lys Asp Pro Val Tyr Thr Pro Met Gly Val Phe 420 425
430 Ile Thr Ala Trp Ala Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys
435 440 445 Tyr Asp Arg Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu
Thr Gly 450 455 460 Thr Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp
Pro Lys Lys Leu 465 470 475 480 Gly Tyr Trp Ala His Glu Ser Thr Phe
Lys Arg Ala Lys Tyr Leu Arg 485 490 495 Gln Lys Thr Tyr Ile Gln Asp
Ile Tyr Met Lys Glu Val Asp Gly Lys 500 505 510 Leu Val Glu Gly Ser
Pro Asp Asp Tyr Thr Asp Ile Lys Phe Ser Val 515 520 525 Lys Cys Ala
Gly Met Thr Asp Lys Ile Lys Lys Glu Val Thr Phe Glu 530 535 540 Asn
Phe Lys Val Gly Phe Ser Arg Lys Met Lys Pro Lys Pro Val Gln 545 550
555 560 Val Pro Gly Gly Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys
Ser 565 570 575 Gly Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly
Gly Gly Ser 580 585 590 Gly Gly Gly Ser Gly Gly Ser Ala Trp Ser His
Pro Gln Phe Glu Lys 595 600 605 10 1390DNAEscherichia coli
10atgatgaacg atggcaaaca gcagagcacc ttcctgtttc atgattatga aaccttcggt
60acccatccgg ccctggatcg tccggcgcag tttgcggcca ttcgcaccga tagcgaattc
120aatgtgattg gcgaaccgga agtgttttat tgcaaaccgg ccgatgatta
tctgccgcag 180ccgggtgcgg tgctgattac cggtattacc ccgcaggaag
cgcgcgcgaa aggtgaaaac 240gaagcggcgt ttgccgcgcg cattcatagc
ctgtttaccg tgccgaaaac ctgcattctg 300ggctataaca atgtgcgctt
cgatgatgaa gttacccgta atatctttta tcgtaacttt 360tatgatccgt
atgcgtggag ctggcagcat gataacagcc gttgggatct gctggatgtg
420atgcgcgcgt gctatgcgct gcgcccggaa ggcattaatt ggccggaaaa
cgatgatggc 480ctgccgagct ttcgtctgga acatctgacc
aaagccaacg gcattgaaca tagcaatgcc 540catgatgcga tggccgatgt
ttatgcgacc attgcgatgg cgaaactggt taaaacccgt 600cagccgcgcc
tgtttgatta tctgtttacc caccgtaaca aacacaaact gatggcgctg
660attgatgttc cgcagatgaa accgctggtg catgtgagcg gcatgtttgg
cgcctggcgc 720ggcaacacca gctgggtggc cccgctggcc tggcacccgg
aaaatcgtaa cgccgtgatt 780atggttgatc tggccggtga tattagcccg
ctgctggaac tggatagcga taccctgcgt 840gaacgcctgt ataccgccaa
aaccgatctg ggcgataatg ccgccgtgcc ggtgaaactg 900gttcacatta
acaaatgccc ggtgctggcc caggcgaaca ccctgcgccc ggaagatgcg
960gatcgtctgg gtattaatcg ccagcattgt ctggataatc tgaaaatcct
gcgtgaaaac 1020ccgcaggtgc gtgaaaaagt ggtggcgatc ttcgcggaag
cggaaccgtt caccccgagc 1080gataacgtgg atgcgcagct gtataacggc
ttctttagcg atgccgatcg cgcggcgatg 1140aaaatcgttc tggaaaccga
accgcgcaat ctgccggcgc tggatattac ctttgttgat 1200aaacgtattg
aaaaactgct gtttaattat cgtgcgcgca attttccggg taccctggat
1260tatgccgaac agcagcgttg gctggaacat cgtcgtcagg ttttcacccc
ggaatttctg 1320cagggttatg cggatgaact gcagatgctg gttcagcagt
atgccgatga taaagaaaaa 1380gtggcgctgc 139011485PRTEscherichia coli
11Met Met Asn Asp Gly Lys Gln Gln Ser Thr Phe Leu Phe His Asp Tyr 1
5 10 15 Glu Thr Phe Gly Thr His Pro Ala Leu Asp Arg Pro Ala Gln Phe
Ala 20 25 30 Ala Ile Arg Thr Asp Ser Glu Phe Asn Val Ile Gly Glu
Pro Glu Val 35 40 45 Phe Tyr Cys Lys Pro Ala Asp Asp Tyr Leu Pro
Gln Pro Gly Ala Val 50 55 60 Leu Ile Thr Gly Ile Thr Pro Gln Glu
Ala Arg Ala Lys Gly Glu Asn 65 70 75 80 Glu Ala Ala Phe Ala Ala Arg
Ile His Ser Leu Phe Thr Val Pro Lys 85 90 95 Thr Cys Ile Leu Gly
Tyr Asn Asn Val Arg Phe Asp Asp Glu Val Thr 100 105 110 Arg Asn Ile
Phe Tyr Arg Asn Phe Tyr Asp Pro Tyr Ala Trp Ser Trp 115 120 125 Gln
His Asp Asn Ser Arg Trp Asp Leu Leu Asp Val Met Arg Ala Cys 130 135
140 Tyr Ala Leu Arg Pro Glu Gly Ile Asn Trp Pro Glu Asn Asp Asp Gly
145 150 155 160 Leu Pro Ser Phe Arg Leu Glu His Leu Thr Lys Ala Asn
Gly Ile Glu 165 170 175 His Ser Asn Ala His Asp Ala Met Ala Asp Val
Tyr Ala Thr Ile Ala 180 185 190 Met Ala Lys Leu Val Lys Thr Arg Gln
Pro Arg Leu Phe Asp Tyr Leu 195 200 205 Phe Thr His Arg Asn Lys His
Lys Leu Met Ala Leu Ile Asp Val Pro 210 215 220 Gln Met Lys Pro Leu
Val His Val Ser Gly Met Phe Gly Ala Trp Arg 225 230 235 240 Gly Asn
Thr Ser Trp Val Ala Pro Leu Ala Trp His Pro Glu Asn Arg 245 250 255
Asn Ala Val Ile Met Val Asp Leu Ala Gly Asp Ile Ser Pro Leu Leu 260
265 270 Glu Leu Asp Ser Asp Thr Leu Arg Glu Arg Leu Tyr Thr Ala Lys
Thr 275 280 285 Asp Leu Gly Asp Asn Ala Ala Val Pro Val Lys Leu Val
His Ile Asn 290 295 300 Lys Cys Pro Val Leu Ala Gln Ala Asn Thr Leu
Arg Pro Glu Asp Ala 305 310 315 320 Asp Arg Leu Gly Ile Asn Arg Gln
His Cys Leu Asp Asn Leu Lys Ile 325 330 335 Leu Arg Glu Asn Pro Gln
Val Arg Glu Lys Val Val Ala Ile Phe Ala 340 345 350 Glu Ala Glu Pro
Phe Thr Pro Ser Asp Asn Val Asp Ala Gln Leu Tyr 355 360 365 Asn Gly
Phe Phe Ser Asp Ala Asp Arg Ala Ala Met Lys Ile Val Leu 370 375 380
Glu Thr Glu Pro Arg Asn Leu Pro Ala Leu Asp Ile Thr Phe Val Asp 385
390 395 400 Lys Arg Ile Glu Lys Leu Leu Phe Asn Tyr Arg Ala Arg Asn
Phe Pro 405 410 415 Gly Thr Leu Asp Tyr Ala Glu Gln Gln Arg Trp Leu
Glu His Arg Arg 420 425 430 Gln Val Phe Thr Pro Glu Phe Leu Gln Gly
Tyr Ala Asp Glu Leu Gln 435 440 445 Met Leu Val Gln Gln Tyr Ala Asp
Asp Lys Glu Lys Val Ala Leu Leu 450 455 460 Lys Ala Leu Trp Gln Tyr
Ala Glu Glu Ile Val Ser Gly Ser Gly His 465 470 475 480 His His His
His His 485 12804DNAEscherichia coli 12atgaaatttg tctcttttaa
tatcaacggc ctgcgcgcca gacctcacca gcttgaagcc 60atcgtcgaaa agcaccaacc
ggatgtgatt ggcctgcagg agacaaaagt tcatgacgat 120atgtttccgc
tcgaagaggt ggcgaagctc ggctacaacg tgttttatca cgggcagaaa
180ggccattatg gcgtggcgct gctgaccaaa gagacgccga ttgccgtgcg
tcgcggcttt 240cccggtgacg acgaagaggc gcagcggcgg attattatgg
cggaaatccc ctcactgctg 300ggtaatgtca ccgtgatcaa cggttacttc
ccgcagggtg aaagccgcga ccatccgata 360aaattcccgg caaaagcgca
gttttatcag aatctgcaaa actacctgga aaccgaactc 420aaacgtgata
atccggtact gattatgggc gatatgaata tcagccctac agatctggat
480atcggcattg gcgaagaaaa ccgtaagcgc tggctgcgta ccggtaaatg
ctctttcctg 540ccggaagagc gcgaatggat ggacaggctg atgagctggg
ggttggtcga taccttccgc 600catgcgaatc cgcaaacagc agatcgtttc
tcatggtttg attaccgctc aaaaggtttt 660gacgataacc gtggtctgcg
catcgacctg ctgctcgcca gccaaccgct ggcagaatgt 720tgcgtagaaa
ccggcatcga ctatgaaatc cgcagcatgg aaaaaccgtc cgatcacgcc
780cccgtctggg cgaccttccg ccgc 80413268PRTEscherichia coli 13Met Lys
Phe Val Ser Phe Asn Ile Asn Gly Leu Arg Ala Arg Pro His 1 5 10 15
Gln Leu Glu Ala Ile Val Glu Lys His Gln Pro Asp Val Ile Gly Leu 20
25 30 Gln Glu Thr Lys Val His Asp Asp Met Phe Pro Leu Glu Glu Val
Ala 35 40 45 Lys Leu Gly Tyr Asn Val Phe Tyr His Gly Gln Lys Gly
His Tyr Gly 50 55 60 Val Ala Leu Leu Thr Lys Glu Thr Pro Ile Ala
Val Arg Arg Gly Phe 65 70 75 80 Pro Gly Asp Asp Glu Glu Ala Gln Arg
Arg Ile Ile Met Ala Glu Ile 85 90 95 Pro Ser Leu Leu Gly Asn Val
Thr Val Ile Asn Gly Tyr Phe Pro Gln 100 105 110 Gly Glu Ser Arg Asp
His Pro Ile Lys Phe Pro Ala Lys Ala Gln Phe 115 120 125 Tyr Gln Asn
Leu Gln Asn Tyr Leu Glu Thr Glu Leu Lys Arg Asp Asn 130 135 140 Pro
Val Leu Ile Met Gly Asp Met Asn Ile Ser Pro Thr Asp Leu Asp 145 150
155 160 Ile Gly Ile Gly Glu Glu Asn Arg Lys Arg Trp Leu Arg Thr Gly
Lys 165 170 175 Cys Ser Phe Leu Pro Glu Glu Arg Glu Trp Met Asp Arg
Leu Met Ser 180 185 190 Trp Gly Leu Val Asp Thr Phe Arg His Ala Asn
Pro Gln Thr Ala Asp 195 200 205 Arg Phe Ser Trp Phe Asp Tyr Arg Ser
Lys Gly Phe Asp Asp Asn Arg 210 215 220 Gly Leu Arg Ile Asp Leu Leu
Leu Ala Ser Gln Pro Leu Ala Glu Cys 225 230 235 240 Cys Val Glu Thr
Gly Ile Asp Tyr Glu Ile Arg Ser Met Glu Lys Pro 245 250 255 Ser Asp
His Ala Pro Val Trp Ala Thr Phe Arg Arg 260 265 141275DNAThermus
thermophilus 14atgtttcgtc gtaaagaaga tctggatccg ccgctggcac
tgctgccgct gaaaggcctg 60cgcgaagccg ccgcactgct ggaagaagcg ctgcgtcaag
gtaaacgcat tcgtgttcac 120ggcgactatg atgcggatgg cctgaccggc
accgcgatcc tggttcgtgg tctggccgcc 180ctgggtgcgg atgttcatcc
gtttatcccg caccgcctgg aagaaggcta tggtgtcctg 240atggaacgcg
tcccggaaca tctggaagcc tcggacctgt ttctgaccgt tgactgcggc
300attaccaacc atgcggaact gcgcgaactg ctggaaaatg gcgtggaagt
cattgttacc 360gatcatcata cgccgggcaa aacgccgccg ccgggtctgg
tcgtgcatcc ggcgctgacg 420ccggatctga aagaaaaacc gaccggcgca
ggcgtggcgt ttctgctgct gtgggcactg 480catgaacgcc tgggcctgcc
gccgccgctg gaatacgcgg acctggcagc cgttggcacc 540attgccgacg
ttgccccgct gtggggttgg aatcgtgcac tggtgaaaga aggtctggca
600cgcatcccgg cttcatcttg ggtgggcctg cgtctgctgg ctgaagccgt
gggctatacc 660ggcaaagcgg tcgaagtcgc tttccgcatc gcgccgcgca
tcaatgcggc ttcccgcctg 720ggcgaagcgg aaaaagccct gcgcctgctg
ctgacggatg atgcggcaga agctcaggcg 780ctggtcggcg aactgcaccg
tctgaacgcc cgtcgtcaga ccctggaaga agcgatgctg 840cgcaaactgc
tgccgcaggc cgacccggaa gcgaaagcca tcgttctgct ggacccggaa
900ggccatccgg gtgttatggg tattgtggcc tctcgcatcc tggaagcgac
cctgcgcccg 960gtctttctgg tggcccaggg caaaggcacc gtgcgttcgc
tggctccgat ttccgccgtc 1020gaagcactgc gcagcgcgga agatctgctg
ctgcgttatg gtggtcataa agaagcggcg 1080ggtttcgcaa tggatgaagc
gctgtttccg gcgttcaaag cacgcgttga agcgtatgcc 1140gcacgtttcc
cggatccggt tcgtgaagtg gcactgctgg atctgctgcc ggaaccgggc
1200ctgctgccgc aggtgttccg tgaactggca ctgctggaac cgtatggtga
aggtaacccg 1260gaaccgctgt tcctg 127515425PRTThermus thermophilus
15Met Phe Arg Arg Lys Glu Asp Leu Asp Pro Pro Leu Ala Leu Leu Pro 1
5 10 15 Leu Lys Gly Leu Arg Glu Ala Ala Ala Leu Leu Glu Glu Ala Leu
Arg 20 25 30 Gln Gly Lys Arg Ile Arg Val His Gly Asp Tyr Asp Ala
Asp Gly Leu 35 40 45 Thr Gly Thr Ala Ile Leu Val Arg Gly Leu Ala
Ala Leu Gly Ala Asp 50 55 60 Val His Pro Phe Ile Pro His Arg Leu
Glu Glu Gly Tyr Gly Val Leu 65 70 75 80 Met Glu Arg Val Pro Glu His
Leu Glu Ala Ser Asp Leu Phe Leu Thr 85 90 95 Val Asp Cys Gly Ile
Thr Asn His Ala Glu Leu Arg Glu Leu Leu Glu 100 105 110 Asn Gly Val
Glu Val Ile Val Thr Asp His His Thr Pro Gly Lys Thr 115 120 125 Pro
Pro Pro Gly Leu Val Val His Pro Ala Leu Thr Pro Asp Leu Lys 130 135
140 Glu Lys Pro Thr Gly Ala Gly Val Ala Phe Leu Leu Leu Trp Ala Leu
145 150 155 160 His Glu Arg Leu Gly Leu Pro Pro Pro Leu Glu Tyr Ala
Asp Leu Ala 165 170 175 Ala Val Gly Thr Ile Ala Asp Val Ala Pro Leu
Trp Gly Trp Asn Arg 180 185 190 Ala Leu Val Lys Glu Gly Leu Ala Arg
Ile Pro Ala Ser Ser Trp Val 195 200 205 Gly Leu Arg Leu Leu Ala Glu
Ala Val Gly Tyr Thr Gly Lys Ala Val 210 215 220 Glu Val Ala Phe Arg
Ile Ala Pro Arg Ile Asn Ala Ala Ser Arg Leu 225 230 235 240 Gly Glu
Ala Glu Lys Ala Leu Arg Leu Leu Leu Thr Asp Asp Ala Ala 245 250 255
Glu Ala Gln Ala Leu Val Gly Glu Leu His Arg Leu Asn Ala Arg Arg 260
265 270 Gln Thr Leu Glu Glu Ala Met Leu Arg Lys Leu Leu Pro Gln Ala
Asp 275 280 285 Pro Glu Ala Lys Ala Ile Val Leu Leu Asp Pro Glu Gly
His Pro Gly 290 295 300 Val Met Gly Ile Val Ala Ser Arg Ile Leu Glu
Ala Thr Leu Arg Pro 305 310 315 320 Val Phe Leu Val Ala Gln Gly Lys
Gly Thr Val Arg Ser Leu Ala Pro 325 330 335 Ile Ser Ala Val Glu Ala
Leu Arg Ser Ala Glu Asp Leu Leu Leu Arg 340 345 350 Tyr Gly Gly His
Lys Glu Ala Ala Gly Phe Ala Met Asp Glu Ala Leu 355 360 365 Phe Pro
Ala Phe Lys Ala Arg Val Glu Ala Tyr Ala Ala Arg Phe Pro 370 375 380
Asp Pro Val Arg Glu Val Ala Leu Leu Asp Leu Leu Pro Glu Pro Gly 385
390 395 400 Leu Leu Pro Gln Val Phe Arg Glu Leu Ala Leu Leu Glu Pro
Tyr Gly 405 410 415 Glu Gly Asn Pro Glu Pro Leu Phe Leu 420 425
16738DNABacteriophage lambda 16tccggaagcg gctctggtag tggttctggc
atgacaccgg acattatcct gcagcgtacc 60gggatcgatg tgagagctgt cgaacagggg
gatgatgcgt ggcacaaatt acggctcggc 120gtcatcaccg cttcagaagt
tcacaacgtg atagcaaaac cccgctccgg aaagaagtgg 180cctgacatga
aaatgtccta cttccacacc ctgcttgctg aggtttgcac cggtgtggct
240ccggaagtta acgctaaagc actggcctgg ggaaaacagt acgagaacga
cgccagaacc 300ctgtttgaat tcacttccgg cgtgaatgtt actgaatccc
cgatcatcta tcgcgacgaa 360agtatgcgta ccgcctgctc tcccgatggt
ttatgcagtg acggcaacgg ccttgaactg 420aaatgcccgt ttacctcccg
ggatttcatg aagttccggc tcggtggttt cgaggccata 480aagtcagctt
acatggccca ggtgcagtac agcatgtggg tgacgcgaaa aaatgcctgg
540tactttgcca actatgaccc gcgtatgaag cgtgaaggcc tgcattatgt
cgtgattgag 600cgggatgaaa agtacatggc gagttttgac gagatcgtgc
cggagttcat cgaaaaaatg 660gacgaggcac tggctgaaat tggttttgta
tttggggagc aatggcgatc tggctctggt 720tccggcagcg gttccgga
73817226PRTBacteriophage lambda 17Met Thr Pro Asp Ile Ile Leu Gln
Arg Thr Gly Ile Asp Val Arg Ala 1 5 10 15 Val Glu Gln Gly Asp Asp
Ala Trp His Lys Leu Arg Leu Gly Val Ile 20 25 30 Thr Ala Ser Glu
Val His Asn Val Ile Ala Lys Pro Arg Ser Gly Lys 35 40 45 Lys Trp
Pro Asp Met Lys Met Ser Tyr Phe His Thr Leu Leu Ala Glu 50 55 60
Val Cys Thr Gly Val Ala Pro Glu Val Asn Ala Lys Ala Leu Ala Trp 65
70 75 80 Gly Lys Gln Tyr Glu Asn Asp Ala Arg Thr Leu Phe Glu Phe
Thr Ser 85 90 95 Gly Val Asn Val Thr Glu Ser Pro Ile Ile Tyr Arg
Asp Glu Ser Met 100 105 110 Arg Thr Ala Cys Ser Pro Asp Gly Leu Cys
Ser Asp Gly Asn Gly Leu 115 120 125 Glu Leu Lys Cys Pro Phe Thr Ser
Arg Asp Phe Met Lys Phe Arg Leu 130 135 140 Gly Gly Phe Glu Ala Ile
Lys Ser Ala Tyr Met Ala Gln Val Gln Tyr 145 150 155 160 Ser Met Trp
Val Thr Arg Lys Asn Ala Trp Tyr Phe Ala Asn Tyr Asp 165 170 175 Pro
Arg Met Lys Arg Glu Gly Leu His Tyr Val Val Ile Glu Arg Asp 180 185
190 Glu Lys Tyr Met Ala Ser Phe Asp Glu Ile Val Pro Glu Phe Ile Glu
195 200 205 Lys Met Asp Glu Ala Leu Ala Glu Ile Gly Phe Val Phe Gly
Glu Gln 210 215 220 Trp Arg 225 18760PRTMethanococcoides burtonii
18Met Met Ile Arg Glu Leu Asp Ile Pro Arg Asp Ile Ile Gly Phe Tyr 1
5 10 15 Glu Asp Ser Gly Ile Lys Glu Leu Tyr Pro Pro Gln Ala Glu Ala
Ile 20 25 30 Glu Met Gly Leu Leu Glu Lys Lys Asn Leu Leu Ala Ala
Ile Pro Thr 35 40 45 Ala Ser Gly Lys Thr Leu Leu Ala Glu Leu Ala
Met Ile Lys Ala Ile 50 55 60 Arg Glu Gly Gly Lys Ala Leu Tyr Ile
Val Pro Leu Arg Ala Leu Ala 65 70 75 80 Ser Glu Lys Phe Glu Arg Phe
Lys Glu Leu Ala Pro Phe Gly Ile Lys 85 90 95 Val Gly Ile Ser Thr
Gly Asp Leu Asp Ser Arg Ala Asp Trp Leu Gly 100 105 110 Val Asn Asp
Ile Ile Val Ala Thr Ser Glu Lys Thr Asp Ser Leu Leu 115 120 125 Arg
Asn Gly Thr Ser Trp Met Asp Glu Ile Thr Thr Val Val Val Asp 130 135
140 Glu Ile His Leu Leu Asp Ser Lys Asn Arg Gly Pro Thr Leu Glu Val
145 150 155 160 Thr Ile Thr Lys Leu Met Arg Leu Asn Pro Asp Val Gln
Val Val Ala 165 170 175 Leu Ser Ala Thr Val Gly Asn Ala Arg Glu Met
Ala Asp Trp Leu Gly 180 185 190 Ala Ala Leu Val Leu Ser Glu Trp Arg
Pro Thr Asp Leu His Glu Gly 195 200 205 Val Leu Phe Gly Asp Ala Ile
Asn Phe Pro Gly Ser Gln Lys Lys Ile 210 215 220 Asp Arg Leu Glu Lys
Asp Asp Ala Val Asn Leu Val Leu Asp Thr Ile 225 230 235 240 Lys Ala
Glu Gly Gln Cys Leu Val Phe Glu Ser Ser Arg Arg Asn Cys 245 250 255
Ala Gly Phe Ala Lys Thr Ala Ser Ser Lys Val Ala Lys Ile Leu Asp 260
265
270 Asn Asp Ile Met Ile Lys Leu Ala Gly Ile Ala Glu Glu Val Glu Ser
275 280 285 Thr Gly Glu Thr Asp Thr Ala Ile Val Leu Ala Asn Cys Ile
Arg Lys 290 295 300 Gly Val Ala Phe His His Ala Gly Leu Asn Ser Asn
His Arg Lys Leu 305 310 315 320 Val Glu Asn Gly Phe Arg Gln Asn Leu
Ile Lys Val Ile Ser Ser Thr 325 330 335 Pro Thr Leu Ala Ala Gly Leu
Asn Leu Pro Ala Arg Arg Val Ile Ile 340 345 350 Arg Ser Tyr Arg Arg
Phe Asp Ser Asn Phe Gly Met Gln Pro Ile Pro 355 360 365 Val Leu Glu
Tyr Lys Gln Met Ala Gly Arg Ala Gly Arg Pro His Leu 370 375 380 Asp
Pro Tyr Gly Glu Ser Val Leu Leu Ala Lys Thr Tyr Asp Glu Phe 385 390
395 400 Ala Gln Leu Met Glu Asn Tyr Val Glu Ala Asp Ala Glu Asp Ile
Trp 405 410 415 Ser Lys Leu Gly Thr Glu Asn Ala Leu Arg Thr His Val
Leu Ser Thr 420 425 430 Ile Val Asn Gly Phe Ala Ser Thr Arg Gln Glu
Leu Phe Asp Phe Phe 435 440 445 Gly Ala Thr Phe Phe Ala Tyr Gln Gln
Asp Lys Trp Met Leu Glu Glu 450 455 460 Val Ile Asn Asp Cys Leu Glu
Phe Leu Ile Asp Lys Ala Met Val Ser 465 470 475 480 Glu Thr Glu Asp
Ile Glu Asp Ala Ser Lys Leu Phe Leu Arg Gly Thr 485 490 495 Arg Leu
Gly Ser Leu Val Ser Met Leu Tyr Ile Asp Pro Leu Ser Gly 500 505 510
Ser Lys Ile Val Asp Gly Phe Lys Asp Ile Gly Lys Ser Thr Gly Gly 515
520 525 Asn Met Gly Ser Leu Glu Asp Asp Lys Gly Asp Asp Ile Thr Val
Thr 530 535 540 Asp Met Thr Leu Leu His Leu Val Cys Ser Thr Pro Asp
Met Arg Gln 545 550 555 560 Leu Tyr Leu Arg Asn Thr Asp Tyr Thr Ile
Val Asn Glu Tyr Ile Val 565 570 575 Ala His Ser Asp Glu Phe His Glu
Ile Pro Asp Lys Leu Lys Glu Thr 580 585 590 Asp Tyr Glu Trp Phe Met
Gly Glu Val Lys Thr Ala Met Leu Leu Glu 595 600 605 Glu Trp Val Thr
Glu Val Ser Ala Glu Asp Ile Thr Arg His Phe Asn 610 615 620 Val Gly
Glu Gly Asp Ile His Ala Leu Ala Asp Thr Ser Glu Trp Leu 625 630 635
640 Met His Ala Ala Ala Lys Leu Ala Glu Leu Leu Gly Val Glu Tyr Ser
645 650 655 Ser His Ala Tyr Ser Leu Glu Lys Arg Ile Arg Tyr Gly Ser
Gly Leu 660 665 670 Asp Leu Met Glu Leu Val Gly Ile Arg Gly Val Gly
Arg Val Arg Ala 675 680 685 Arg Lys Leu Tyr Asn Ala Gly Phe Val Ser
Val Ala Lys Leu Lys Gly 690 695 700 Ala Asp Ile Ser Val Leu Ser Lys
Leu Val Gly Pro Lys Val Ala Tyr 705 710 715 720 Asn Ile Leu Ser Gly
Ile Gly Val Arg Val Asn Asp Lys His Phe Asn 725 730 735 Ser Ala Pro
Ile Ser Ser Asn Thr Leu Asp Thr Leu Leu Asp Lys Asn 740 745 750 Gln
Lys Thr Phe Asn Asp Phe Gln 755 760 19707PRTCenarchaeum symbiosum
19Met Arg Ile Ser Glu Leu Asp Ile Pro Arg Pro Ala Ile Glu Phe Leu 1
5 10 15 Glu Gly Glu Gly Tyr Lys Lys Leu Tyr Pro Pro Gln Ala Ala Ala
Ala 20 25 30 Lys Ala Gly Leu Thr Asp Gly Lys Ser Val Leu Val Ser
Ala Pro Thr 35 40 45 Ala Ser Gly Lys Thr Leu Ile Ala Ala Ile Ala
Met Ile Ser His Leu 50 55 60 Ser Arg Asn Arg Gly Lys Ala Val Tyr
Leu Ser Pro Leu Arg Ala Leu 65 70 75 80 Ala Ala Glu Lys Phe Ala Glu
Phe Gly Lys Ile Gly Gly Ile Pro Leu 85 90 95 Gly Arg Pro Val Arg
Val Gly Val Ser Thr Gly Asp Phe Glu Lys Ala 100 105 110 Gly Arg Ser
Leu Gly Asn Asn Asp Ile Leu Val Leu Thr Asn Glu Arg 115 120 125 Met
Asp Ser Leu Ile Arg Arg Arg Pro Asp Trp Met Asp Glu Val Gly 130 135
140 Leu Val Ile Ala Asp Glu Ile His Leu Ile Gly Asp Arg Ser Arg Gly
145 150 155 160 Pro Thr Leu Glu Met Val Leu Thr Lys Leu Arg Gly Leu
Arg Ser Ser 165 170 175 Pro Gln Val Val Ala Leu Ser Ala Thr Ile Ser
Asn Ala Asp Glu Ile 180 185 190 Ala Gly Trp Leu Asp Cys Thr Leu Val
His Ser Thr Trp Arg Pro Val 195 200 205 Pro Leu Ser Glu Gly Val Tyr
Gln Asp Gly Glu Val Ala Met Gly Asp 210 215 220 Gly Ser Arg His Glu
Val Ala Ala Thr Gly Gly Gly Pro Ala Val Asp 225 230 235 240 Leu Ala
Ala Glu Ser Val Ala Glu Gly Gly Gln Ser Leu Ile Phe Ala 245 250 255
Asp Thr Arg Ala Arg Ser Ala Ser Leu Ala Ala Lys Ala Ser Ala Val 260
265 270 Ile Pro Glu Ala Lys Gly Ala Asp Ala Ala Lys Leu Ala Ala Ala
Ala 275 280 285 Lys Lys Ile Ile Ser Ser Gly Gly Glu Thr Lys Leu Ala
Lys Thr Leu 290 295 300 Ala Glu Leu Val Glu Lys Gly Ala Ala Phe His
His Ala Gly Leu Asn 305 310 315 320 Gln Asp Cys Arg Ser Val Val Glu
Glu Glu Phe Arg Ser Gly Arg Ile 325 330 335 Arg Leu Leu Ala Ser Thr
Pro Thr Leu Ala Ala Gly Val Asn Leu Pro 340 345 350 Ala Arg Arg Val
Val Ile Ser Ser Val Met Arg Tyr Asn Ser Ser Ser 355 360 365 Gly Met
Ser Glu Pro Ile Ser Ile Leu Glu Tyr Lys Gln Leu Cys Gly 370 375 380
Arg Ala Gly Arg Pro Gln Tyr Asp Lys Ser Gly Glu Ala Ile Val Val 385
390 395 400 Gly Gly Val Asn Ala Asp Glu Ile Phe Asp Arg Tyr Ile Gly
Gly Glu 405 410 415 Pro Glu Pro Ile Arg Ser Ala Met Val Asp Asp Arg
Ala Leu Arg Ile 420 425 430 His Val Leu Ser Leu Val Thr Thr Ser Pro
Gly Ile Lys Glu Asp Asp 435 440 445 Val Thr Glu Phe Phe Leu Gly Thr
Leu Gly Gly Gln Gln Ser Gly Glu 450 455 460 Ser Thr Val Lys Phe Ser
Val Ala Val Ala Leu Arg Phe Leu Gln Glu 465 470 475 480 Glu Gly Met
Leu Gly Arg Arg Gly Gly Arg Leu Ala Ala Thr Lys Met 485 490 495 Gly
Arg Leu Val Ser Arg Leu Tyr Met Asp Pro Met Thr Ala Val Thr 500 505
510 Leu Arg Asp Ala Val Gly Glu Ala Ser Pro Gly Arg Met His Thr Leu
515 520 525 Gly Phe Leu His Leu Val Ser Glu Cys Ser Glu Phe Met Pro
Arg Phe 530 535 540 Ala Leu Arg Gln Lys Asp His Glu Val Ala Glu Met
Met Leu Glu Ala 545 550 555 560 Gly Arg Gly Glu Leu Leu Arg Pro Val
Tyr Ser Tyr Glu Cys Gly Arg 565 570 575 Gly Leu Leu Ala Leu His Arg
Trp Ile Gly Glu Ser Pro Glu Ala Lys 580 585 590 Leu Ala Glu Asp Leu
Lys Phe Glu Ser Gly Asp Val His Arg Met Val 595 600 605 Glu Ser Ser
Gly Trp Leu Leu Arg Cys Ile Trp Glu Ile Ser Lys His 610 615 620 Gln
Glu Arg Pro Asp Leu Leu Gly Glu Leu Asp Val Leu Arg Ser Arg 625 630
635 640 Val Ala Tyr Gly Ile Lys Ala Glu Leu Val Pro Leu Val Ser Ile
Lys 645 650 655 Gly Ile Gly Arg Val Arg Ser Arg Arg Leu Phe Arg Gly
Gly Ile Lys 660 665 670 Gly Pro Gly Asp Leu Ala Ala Val Pro Val Glu
Arg Leu Ser Arg Val 675 680 685 Glu Gly Ile Gly Ala Thr Leu Ala Asn
Asn Ile Lys Ser Gln Leu Arg 690 695 700 Lys Gly Gly 705
20720PRTThermococcus gammatolerans 20Met Lys Val Asp Glu Leu Pro
Val Asp Glu Arg Leu Lys Ala Val Leu 1 5 10 15 Lys Glu Arg Gly Ile
Glu Glu Leu Tyr Pro Pro Gln Ala Glu Ala Leu 20 25 30 Lys Ser Gly
Ala Leu Glu Gly Arg Asn Leu Val Leu Ala Ile Pro Thr 35 40 45 Ala
Ser Gly Lys Thr Leu Val Ser Glu Ile Val Met Val Asn Lys Leu 50 55
60 Ile Gln Glu Gly Gly Lys Ala Val Tyr Leu Val Pro Leu Lys Ala Leu
65 70 75 80 Ala Glu Glu Lys Tyr Arg Glu Phe Lys Glu Trp Glu Lys Leu
Gly Leu 85 90 95 Lys Val Ala Ala Thr Thr Gly Asp Tyr Asp Ser Thr
Asp Asp Trp Leu 100 105 110 Gly Arg Tyr Asp Ile Ile Val Ala Thr Ala
Glu Lys Phe Asp Ser Leu 115 120 125 Leu Arg His Gly Ala Arg Trp Ile
Asn Asp Val Lys Leu Val Val Ala 130 135 140 Asp Glu Val His Leu Ile
Gly Ser Tyr Asp Arg Gly Ala Thr Leu Glu 145 150 155 160 Met Ile Leu
Thr His Met Leu Gly Arg Ala Gln Ile Leu Ala Leu Ser 165 170 175 Ala
Thr Val Gly Asn Ala Glu Glu Leu Ala Glu Trp Leu Asp Ala Ser 180 185
190 Leu Val Val Ser Asp Trp Arg Pro Val Gln Leu Arg Arg Gly Val Phe
195 200 205 His Leu Gly Thr Leu Ile Trp Glu Asp Gly Lys Val Glu Ser
Tyr Pro 210 215 220 Glu Asn Trp Tyr Ser Leu Val Val Asp Ala Val Lys
Arg Gly Lys Gly 225 230 235 240 Ala Leu Val Phe Val Asn Thr Arg Arg
Ser Ala Glu Lys Glu Ala Leu 245 250 255 Ala Leu Ser Lys Leu Val Ser
Ser His Leu Thr Lys Pro Glu Lys Arg 260 265 270 Ala Leu Glu Ser Leu
Ala Ser Gln Leu Glu Asp Asn Pro Thr Ser Glu 275 280 285 Lys Leu Lys
Arg Ala Leu Arg Gly Gly Val Ala Phe His His Ala Gly 290 295 300 Leu
Ser Arg Val Glu Arg Thr Leu Ile Glu Asp Ala Phe Arg Glu Gly 305 310
315 320 Leu Ile Lys Val Ile Thr Ala Thr Pro Thr Leu Ser Ala Gly Val
Asn 325 330 335 Leu Pro Ser Phe Arg Val Ile Ile Arg Asp Thr Lys Arg
Tyr Ala Gly 340 345 350 Phe Gly Trp Thr Asp Ile Pro Val Leu Glu Ile
Gln Gln Met Met Gly 355 360 365 Arg Ala Gly Arg Pro Arg Tyr Asp Lys
Tyr Gly Glu Ala Ile Ile Val 370 375 380 Ala Arg Thr Asp Glu Pro Gly
Lys Leu Met Glu Arg Tyr Ile Arg Gly 385 390 395 400 Lys Pro Glu Lys
Leu Phe Ser Met Leu Ala Asn Glu Gln Ala Phe Arg 405 410 415 Ser Gln
Val Leu Ala Leu Ile Thr Asn Phe Gly Ile Arg Ser Phe Pro 420 425 430
Glu Leu Val Arg Phe Leu Glu Arg Thr Phe Tyr Ala His Gln Arg Lys 435
440 445 Asp Leu Ser Ser Leu Glu Tyr Lys Ala Lys Glu Val Val Tyr Phe
Leu 450 455 460 Ile Glu Asn Glu Phe Ile Asp Leu Asp Leu Glu Asp Arg
Phe Ile Pro 465 470 475 480 Leu Pro Phe Gly Lys Arg Thr Ser Gln Leu
Tyr Ile Asp Pro Leu Thr 485 490 495 Ala Lys Lys Phe Lys Asp Ala Phe
Pro Ala Ile Glu Arg Asn Pro Asn 500 505 510 Pro Phe Gly Ile Phe Gln
Leu Ile Ala Ser Thr Pro Asp Met Ala Thr 515 520 525 Leu Thr Ala Arg
Arg Arg Glu Met Glu Asp Tyr Leu Asp Leu Ala Tyr 530 535 540 Glu Leu
Glu Asp Lys Leu Tyr Ala Ser Ile Pro Tyr Tyr Glu Asp Ser 545 550 555
560 Arg Phe Gln Gly Phe Leu Gly Gln Val Lys Thr Ala Lys Val Leu Leu
565 570 575 Asp Trp Ile Asn Glu Val Pro Glu Ala Arg Ile Tyr Glu Thr
Tyr Ser 580 585 590 Ile Asp Pro Gly Asp Leu Tyr Arg Leu Leu Glu Leu
Ala Asp Trp Leu 595 600 605 Met Tyr Ser Leu Ile Glu Leu Tyr Lys Leu
Phe Glu Pro Lys Glu Glu 610 615 620 Ile Leu Asn Tyr Leu Arg Asp Leu
His Leu Arg Leu Arg His Gly Val 625 630 635 640 Arg Glu Glu Leu Leu
Glu Leu Val Arg Leu Pro Asn Ile Gly Arg Lys 645 650 655 Arg Ala Arg
Ala Leu Tyr Asn Ala Gly Phe Arg Ser Val Glu Ala Ile 660 665 670 Ala
Asn Ala Lys Pro Ala Glu Leu Leu Ala Val Glu Gly Ile Gly Ala 675 680
685 Lys Ile Leu Asp Gly Ile Tyr Arg His Leu Gly Ile Glu Lys Arg Val
690 695 700 Thr Glu Glu Lys Pro Lys Arg Lys Gly Thr Leu Glu Asp Phe
Leu Arg 705 710 715 720 21799PRTMethanospirillum hungatei 21Met Glu
Ile Ala Ser Leu Pro Leu Pro Asp Ser Phe Ile Arg Ala Cys 1 5 10 15
His Ala Lys Gly Ile Arg Ser Leu Tyr Pro Pro Gln Ala Glu Cys Ile 20
25 30 Glu Lys Gly Leu Leu Glu Gly Lys Asn Leu Leu Ile Ser Ile Pro
Thr 35 40 45 Ala Ser Gly Lys Thr Leu Leu Ala Glu Met Ala Met Trp
Ser Arg Ile 50 55 60 Ala Ala Gly Gly Lys Cys Leu Tyr Ile Val Pro
Leu Arg Ala Leu Ala 65 70 75 80 Ser Glu Lys Tyr Asp Glu Phe Ser Lys
Lys Gly Val Ile Arg Val Gly 85 90 95 Ile Ala Thr Gly Asp Leu Asp
Arg Thr Asp Ala Tyr Leu Gly Glu Asn 100 105 110 Asp Ile Ile Val Ala
Thr Ser Glu Lys Thr Asp Ser Leu Leu Arg Asn 115 120 125 Arg Thr Pro
Trp Leu Ser Gln Ile Thr Cys Ile Val Leu Asp Glu Val 130 135 140 His
Leu Ile Gly Ser Glu Asn Arg Gly Ala Thr Leu Glu Met Val Ile 145 150
155 160 Thr Lys Leu Arg Tyr Thr Asn Pro Val Met Gln Ile Ile Gly Leu
Ser 165 170 175 Ala Thr Ile Gly Asn Pro Ala Gln Leu Ala Glu Trp Leu
Asp Ala Thr 180 185 190 Leu Ile Thr Ser Thr Trp Arg Pro Val Asp Leu
Arg Gln Gly Val Tyr 195 200 205 Tyr Asn Gly Lys Ile Arg Phe Ser Asp
Ser Glu Arg Pro Ile Gln Gly 210 215 220 Lys Thr Lys His Asp Asp Leu
Asn Leu Cys Leu Asp Thr Ile Glu Glu 225 230 235 240 Gly Gly Gln Cys
Leu Val Phe Val Ser Ser Arg Arg Asn Ala Glu Gly 245 250 255 Phe Ala
Lys Lys Ala Ala Gly Ala Leu Lys Ala Gly Ser Pro Asp Ser 260 265 270
Lys Ala Leu Ala Gln Glu Leu Arg Arg Leu Arg Asp Arg Asp Glu Gly 275
280 285 Asn Val Leu Ala Asp Cys Val Glu Arg Gly Ala Ala Phe His His
Ala 290 295 300 Gly Leu Ile Arg Gln Glu Arg Thr Ile Ile Glu Glu Gly
Phe Arg Asn 305 310 315 320 Gly Tyr Ile Glu Val Ile Ala Ala Thr Pro
Thr Leu Ala Ala Gly Leu 325 330 335 Asn Leu Pro Ala Arg Arg Val Ile
Ile Arg Asp Tyr Asn Arg Phe Ala 340 345 350 Ser Gly Leu Gly Met Val
Pro Ile Pro Val Gly Glu Tyr His Gln Met 355 360 365 Ala Gly Arg Ala
Gly Arg Pro His Leu Asp Pro Tyr Gly Glu Ala Val 370
375 380 Leu Leu Ala Lys Asp Ala Pro Ser Val Glu Arg Leu Phe Glu Thr
Phe 385 390 395 400 Ile Asp Ala Glu Ala Glu Arg Val Asp Ser Gln Cys
Val Asp Asp Ala 405 410 415 Ser Leu Cys Ala His Ile Leu Ser Leu Ile
Ala Thr Gly Phe Ala His 420 425 430 Asp Gln Glu Ala Leu Ser Ser Phe
Met Glu Arg Thr Phe Tyr Phe Phe 435 440 445 Gln His Pro Lys Thr Arg
Ser Leu Pro Arg Leu Val Ala Asp Ala Ile 450 455 460 Arg Phe Leu Thr
Thr Ala Gly Met Val Glu Glu Arg Glu Asn Thr Leu 465 470 475 480 Ser
Ala Thr Arg Leu Gly Ser Leu Val Ser Arg Leu Tyr Leu Asn Pro 485 490
495 Cys Thr Ala Arg Leu Ile Leu Asp Ser Leu Lys Ser Cys Lys Thr Pro
500 505 510 Thr Leu Ile Gly Leu Leu His Val Ile Cys Val Ser Pro Asp
Met Gln 515 520 525 Arg Leu Tyr Leu Lys Ala Ala Asp Thr Gln Leu Leu
Arg Thr Phe Leu 530 535 540 Phe Lys His Lys Asp Asp Leu Ile Leu Pro
Leu Pro Phe Glu Gln Glu 545 550 555 560 Glu Glu Glu Leu Trp Leu Ser
Gly Leu Lys Thr Ala Leu Val Leu Thr 565 570 575 Asp Trp Ala Asp Glu
Phe Ser Glu Gly Met Ile Glu Glu Arg Tyr Gly 580 585 590 Ile Gly Ala
Gly Asp Leu Tyr Asn Ile Val Asp Ser Gly Lys Trp Leu 595 600 605 Leu
His Gly Thr Glu Arg Leu Val Ser Val Glu Met Pro Glu Met Ser 610 615
620 Gln Val Val Lys Thr Leu Ser Val Arg Val His His Gly Val Lys Ser
625 630 635 640 Glu Leu Leu Pro Leu Val Ala Leu Arg Asn Ile Gly Arg
Val Arg Ala 645 650 655 Arg Thr Leu Tyr Asn Ala Gly Tyr Pro Asp Pro
Glu Ala Val Ala Arg 660 665 670 Ala Gly Leu Ser Thr Ile Ala Arg Ile
Ile Gly Glu Gly Ile Ala Arg 675 680 685 Gln Val Ile Asp Glu Ile Thr
Gly Val Lys Arg Ser Gly Ile His Ser 690 695 700 Ser Asp Asp Asp Tyr
Gln Gln Lys Thr Pro Glu Leu Leu Thr Asp Ile 705 710 715 720 Pro Gly
Ile Gly Lys Lys Met Ala Glu Lys Leu Gln Asn Ala Gly Ile 725 730 735
Ile Thr Val Ser Asp Leu Leu Thr Ala Asp Glu Val Leu Leu Ser Asp 740
745 750 Val Leu Gly Ala Ala Arg Ala Arg Lys Val Leu Ala Phe Leu Ser
Asn 755 760 765 Ser Glu Lys Glu Asn Ser Ser Ser Asp Lys Thr Glu Glu
Ile Pro Asp 770 775 780 Thr Gln Lys Ile Arg Gly Gln Ser Ser Trp Glu
Asp Phe Gly Cys 785 790 795 221756PRTEscherichia coli 22Met Met Ser
Ile Ala Gln Val Arg Ser Ala Gly Ser Ala Gly Asn Tyr 1 5 10 15 Tyr
Thr Asp Lys Asp Asn Tyr Tyr Val Leu Gly Ser Met Gly Glu Arg 20 25
30 Trp Ala Gly Lys Gly Ala Glu Gln Leu Gly Leu Gln Gly Ser Val Asp
35 40 45 Lys Asp Val Phe Thr Arg Leu Leu Glu Gly Arg Leu Pro Asp
Gly Ala 50 55 60 Asp Leu Ser Arg Met Gln Asp Gly Ser Asn Lys His
Arg Pro Gly Tyr 65 70 75 80 Asp Leu Thr Phe Ser Ala Pro Lys Ser Val
Ser Met Met Ala Met Leu 85 90 95 Gly Gly Asp Lys Arg Leu Ile Asp
Ala His Asn Gln Ala Val Asp Phe 100 105 110 Ala Val Arg Gln Val Glu
Ala Leu Ala Ser Thr Arg Val Met Thr Asp 115 120 125 Gly Gln Ser Glu
Thr Val Leu Thr Gly Asn Leu Val Met Ala Leu Phe 130 135 140 Asn His
Asp Thr Ser Arg Asp Gln Glu Pro Gln Leu His Thr His Ala 145 150 155
160 Val Val Ala Asn Val Thr Gln His Asn Gly Glu Trp Lys Thr Leu Ser
165 170 175 Ser Asp Lys Val Gly Lys Thr Gly Phe Ile Glu Asn Val Tyr
Ala Asn 180 185 190 Gln Ile Ala Phe Gly Arg Leu Tyr Arg Glu Lys Leu
Lys Glu Gln Val 195 200 205 Glu Ala Leu Gly Tyr Glu Thr Glu Val Val
Gly Lys His Gly Met Trp 210 215 220 Glu Met Pro Gly Val Pro Val Glu
Ala Phe Ser Gly Arg Ser Gln Ala 225 230 235 240 Ile Arg Glu Ala Val
Gly Glu Asp Ala Ser Leu Lys Ser Arg Asp Val 245 250 255 Ala Ala Leu
Asp Thr Arg Lys Ser Lys Gln His Val Asp Pro Glu Ile 260 265 270 Arg
Met Ala Glu Trp Met Gln Thr Leu Lys Glu Thr Gly Phe Asp Ile 275 280
285 Arg Ala Tyr Arg Asp Ala Ala Asp Gln Arg Thr Glu Ile Arg Thr Gln
290 295 300 Ala Pro Gly Pro Ala Ser Gln Asp Gly Pro Asp Val Gln Gln
Ala Val 305 310 315 320 Thr Gln Ala Ile Ala Gly Leu Ser Glu Arg Lys
Val Gln Phe Thr Tyr 325 330 335 Thr Asp Val Leu Ala Arg Thr Val Gly
Ile Leu Pro Pro Glu Asn Gly 340 345 350 Val Ile Glu Arg Ala Arg Ala
Gly Ile Asp Glu Ala Ile Ser Arg Glu 355 360 365 Gln Leu Ile Pro Leu
Asp Arg Glu Lys Gly Leu Phe Thr Ser Gly Ile 370 375 380 His Val Leu
Asp Glu Leu Ser Val Arg Ala Leu Ser Arg Asp Ile Met 385 390 395 400
Lys Gln Asn Arg Val Thr Val His Pro Glu Lys Ser Val Pro Arg Thr 405
410 415 Ala Gly Tyr Ser Asp Ala Val Ser Val Leu Ala Gln Asp Arg Pro
Ser 420 425 430 Leu Ala Ile Val Ser Gly Gln Gly Gly Ala Ala Gly Gln
Arg Glu Arg 435 440 445 Val Ala Glu Leu Val Met Met Ala Arg Glu Gln
Gly Arg Glu Val Gln 450 455 460 Ile Ile Ala Ala Asp Arg Arg Ser Gln
Met Asn Leu Lys Gln Asp Glu 465 470 475 480 Arg Leu Ser Gly Glu Leu
Ile Thr Gly Arg Arg Gln Leu Leu Glu Gly 485 490 495 Met Ala Phe Thr
Pro Gly Ser Thr Val Ile Val Asp Gln Gly Glu Lys 500 505 510 Leu Ser
Leu Lys Glu Thr Leu Thr Leu Leu Asp Gly Ala Ala Arg His 515 520 525
Asn Val Gln Val Leu Ile Thr Asp Ser Gly Gln Arg Thr Gly Thr Gly 530
535 540 Ser Ala Leu Met Ala Met Lys Asp Ala Gly Val Asn Thr Tyr Arg
Trp 545 550 555 560 Gln Gly Gly Glu Gln Arg Pro Ala Thr Ile Ile Ser
Glu Pro Asp Arg 565 570 575 Asn Val Arg Tyr Ala Arg Leu Ala Gly Asp
Phe Ala Ala Ser Val Lys 580 585 590 Ala Gly Glu Glu Ser Val Ala Gln
Val Ser Gly Val Arg Glu Gln Ala 595 600 605 Ile Leu Thr Gln Ala Ile
Arg Ser Glu Leu Lys Thr Gln Gly Val Leu 610 615 620 Gly His Pro Glu
Val Thr Met Thr Ala Leu Ser Pro Val Trp Leu Asp 625 630 635 640 Ser
Arg Ser Arg Tyr Leu Arg Asp Met Tyr Arg Pro Gly Met Val Met 645 650
655 Glu Gln Trp Asn Pro Glu Thr Arg Ser His Asp Arg Tyr Val Ile Asp
660 665 670 Arg Val Thr Ala Gln Ser His Ser Leu Thr Leu Arg Asp Ala
Gln Gly 675 680 685 Glu Thr Gln Val Val Arg Ile Ser Ser Leu Asp Ser
Ser Trp Ser Leu 690 695 700 Phe Arg Pro Glu Lys Met Pro Val Ala Asp
Gly Glu Arg Leu Arg Val 705 710 715 720 Thr Gly Lys Ile Pro Gly Leu
Arg Val Ser Gly Gly Asp Arg Leu Gln 725 730 735 Val Ala Ser Val Ser
Glu Asp Ala Met Thr Val Val Val Pro Gly Arg 740 745 750 Ala Glu Pro
Ala Ser Leu Pro Val Ser Asp Ser Pro Phe Thr Ala Leu 755 760 765 Lys
Leu Glu Asn Gly Trp Val Glu Thr Pro Gly His Ser Val Ser Asp 770 775
780 Ser Ala Thr Val Phe Ala Ser Val Thr Gln Met Ala Met Asp Asn Ala
785 790 795 800 Thr Leu Asn Gly Leu Ala Arg Ser Gly Arg Asp Val Arg
Leu Tyr Ser 805 810 815 Ser Leu Asp Glu Thr Arg Thr Ala Glu Lys Leu
Ala Arg His Pro Ser 820 825 830 Phe Thr Val Val Ser Glu Gln Ile Lys
Ala Arg Ala Gly Glu Thr Leu 835 840 845 Leu Glu Thr Ala Ile Ser Leu
Gln Lys Ala Gly Leu His Thr Pro Ala 850 855 860 Gln Gln Ala Ile His
Leu Ala Leu Pro Val Leu Glu Ser Lys Asn Leu 865 870 875 880 Ala Phe
Ser Met Val Asp Leu Leu Thr Glu Ala Lys Ser Phe Ala Ala 885 890 895
Glu Gly Thr Gly Phe Thr Glu Leu Gly Gly Glu Ile Asn Ala Gln Ile 900
905 910 Lys Arg Gly Asp Leu Leu Tyr Val Asp Val Ala Lys Gly Tyr Gly
Thr 915 920 925 Gly Leu Leu Val Ser Arg Ala Ser Tyr Glu Ala Glu Lys
Ser Ile Leu 930 935 940 Arg His Ile Leu Glu Gly Lys Glu Ala Val Thr
Pro Leu Met Glu Arg 945 950 955 960 Val Pro Gly Glu Leu Met Glu Thr
Leu Thr Ser Gly Gln Arg Ala Ala 965 970 975 Thr Arg Met Ile Leu Glu
Thr Ser Asp Arg Phe Thr Val Val Gln Gly 980 985 990 Tyr Ala Gly Val
Gly Lys Thr Thr Gln Phe Arg Ala Val Met Ser Ala 995 1000 1005 Val
Asn Met Leu Pro Ala Ser Glu Arg Pro Arg Val Val Gly Leu 1010 1015
1020 Gly Pro Thr His Arg Ala Val Gly Glu Met Arg Ser Ala Gly Val
1025 1030 1035 Asp Ala Gln Thr Leu Ala Ser Phe Leu His Asp Thr Gln
Leu Gln 1040 1045 1050 Gln Arg Ser Gly Glu Thr Pro Asp Phe Ser Asn
Thr Leu Phe Leu 1055 1060 1065 Leu Asp Glu Ser Ser Met Val Gly Asn
Thr Glu Met Ala Arg Ala 1070 1075 1080 Tyr Ala Leu Ile Ala Ala Gly
Gly Gly Arg Ala Val Ala Ser Gly 1085 1090 1095 Asp Thr Asp Gln Leu
Gln Ala Ile Ala Pro Gly Gln Ser Phe Arg 1100 1105 1110 Leu Gln Gln
Thr Arg Ser Ala Ala Asp Val Val Ile Met Lys Glu 1115 1120 1125 Ile
Val Arg Gln Thr Pro Glu Leu Arg Glu Ala Val Tyr Ser Leu 1130 1135
1140 Ile Asn Arg Asp Val Glu Arg Ala Leu Ser Gly Leu Glu Ser Val
1145 1150 1155 Lys Pro Ser Gln Val Pro Arg Leu Glu Gly Ala Trp Ala
Pro Glu 1160 1165 1170 His Ser Val Thr Glu Phe Ser His Ser Gln Glu
Ala Lys Leu Ala 1175 1180 1185 Glu Ala Gln Gln Lys Ala Met Leu Lys
Gly Glu Ala Phe Pro Asp 1190 1195 1200 Ile Pro Met Thr Leu Tyr Glu
Ala Ile Val Arg Asp Tyr Thr Gly 1205 1210 1215 Arg Thr Pro Glu Ala
Arg Glu Gln Thr Leu Ile Val Thr His Leu 1220 1225 1230 Asn Glu Asp
Arg Arg Val Leu Asn Ser Met Ile His Asp Ala Arg 1235 1240 1245 Glu
Lys Ala Gly Glu Leu Gly Lys Glu Gln Val Met Val Pro Val 1250 1255
1260 Leu Asn Thr Ala Asn Ile Arg Asp Gly Glu Leu Arg Arg Leu Ser
1265 1270 1275 Thr Trp Glu Lys Asn Pro Asp Ala Leu Ala Leu Val Asp
Asn Val 1280 1285 1290 Tyr His Arg Ile Ala Gly Ile Ser Lys Asp Asp
Gly Leu Ile Thr 1295 1300 1305 Leu Gln Asp Ala Glu Gly Asn Thr Arg
Leu Ile Ser Pro Arg Glu 1310 1315 1320 Ala Val Ala Glu Gly Val Thr
Leu Tyr Thr Pro Asp Lys Ile Arg 1325 1330 1335 Val Gly Thr Gly Asp
Arg Met Arg Phe Thr Lys Ser Asp Arg Glu 1340 1345 1350 Arg Gly Tyr
Val Ala Asn Ser Val Trp Thr Val Thr Ala Val Ser 1355 1360 1365 Gly
Asp Ser Val Thr Leu Ser Asp Gly Gln Gln Thr Arg Val Ile 1370 1375
1380 Arg Pro Gly Gln Glu Arg Ala Glu Gln His Ile Asp Leu Ala Tyr
1385 1390 1395 Ala Ile Thr Ala His Gly Ala Gln Gly Ala Ser Glu Thr
Phe Ala 1400 1405 1410 Ile Ala Leu Glu Gly Thr Glu Gly Asn Arg Lys
Leu Met Ala Gly 1415 1420 1425 Phe Glu Ser Ala Tyr Val Ala Leu Ser
Arg Met Lys Gln His Val 1430 1435 1440 Gln Val Tyr Thr Asp Asn Arg
Gln Gly Trp Thr Asp Ala Ile Asn 1445 1450 1455 Asn Ala Val Gln Lys
Gly Thr Ala His Asp Val Leu Glu Pro Lys 1460 1465 1470 Pro Asp Arg
Glu Val Met Asn Ala Gln Arg Leu Phe Ser Thr Ala 1475 1480 1485 Arg
Glu Leu Arg Asp Val Ala Ala Gly Arg Ala Val Leu Arg Gln 1490 1495
1500 Ala Gly Leu Ala Gly Gly Asp Ser Pro Ala Arg Phe Ile Ala Pro
1505 1510 1515 Gly Arg Lys Tyr Pro Gln Pro Tyr Val Ala Leu Pro Ala
Phe Asp 1520 1525 1530 Arg Asn Gly Lys Ser Ala Gly Ile Trp Leu Asn
Pro Leu Thr Thr 1535 1540 1545 Asp Asp Gly Asn Gly Leu Arg Gly Phe
Ser Gly Glu Gly Arg Val 1550 1555 1560 Lys Gly Ser Gly Asp Ala Gln
Phe Val Ala Leu Gln Gly Ser Arg 1565 1570 1575 Asn Gly Glu Ser Leu
Leu Ala Asp Asn Met Gln Asp Gly Val Arg 1580 1585 1590 Ile Ala Arg
Asp Asn Pro Asp Ser Gly Val Val Val Arg Ile Ala 1595 1600 1605 Gly
Glu Gly Arg Pro Trp Asn Pro Gly Ala Ile Thr Gly Gly Arg 1610 1615
1620 Val Trp Gly Asp Ile Pro Asp Asn Ser Val Gln Pro Gly Ala Gly
1625 1630 1635 Asn Gly Glu Pro Val Thr Ala Glu Val Leu Ala Gln Arg
Gln Ala 1640 1645 1650 Glu Glu Ala Ile Arg Arg Glu Thr Glu Arg Arg
Ala Asp Glu Ile 1655 1660 1665 Val Arg Lys Met Ala Glu Asn Lys Pro
Asp Leu Pro Asp Gly Lys 1670 1675 1680 Thr Glu Leu Ala Val Arg Asp
Ile Ala Gly Gln Glu Arg Asp Arg 1685 1690 1695 Ser Ala Ile Ser Glu
Arg Glu Thr Ala Leu Pro Glu Ser Val Leu 1700 1705 1710 Arg Glu Ser
Gln Arg Glu Arg Glu Ala Val Arg Glu Val Ala Arg 1715 1720 1725 Glu
Asn Leu Leu Gln Glu Arg Leu Gln Gln Met Glu Arg Asp Met 1730 1735
1740 Val Arg Asp Leu Gln Lys Glu Lys Thr Leu Gly Gly Asp 1745 1750
1755 23726PRTMethanococcoides burtonii 23Met Ser Asp Lys Pro Ala
Phe Met Lys Tyr Phe Thr Gln Ser Ser Cys 1 5 10 15 Tyr Pro Asn Gln
Gln Glu Ala Met Asp Arg Ile His Ser Ala Leu Met 20 25 30 Gln Gln
Gln Leu Val Leu Phe Glu Gly Ala Cys Gly Thr Gly Lys Thr 35 40 45
Leu Ser Ala Leu Val Pro Ala Leu His Val Gly Lys Met Leu Gly Lys 50
55 60 Thr Val Ile Ile Ala Thr Asn Val His Gln Gln Met Val Gln Phe
Ile 65 70 75
80 Asn Glu Ala Arg Asp Ile Lys Lys Val Gln Asp Val Lys Val Ala Val
85 90 95 Ile Lys Gly Lys Thr Ala Met Cys Pro Gln Glu Ala Asp Tyr
Glu Glu 100 105 110 Cys Ser Val Lys Arg Glu Asn Thr Phe Glu Leu Met
Glu Thr Glu Arg 115 120 125 Glu Ile Tyr Leu Lys Arg Gln Glu Leu Asn
Ser Ala Arg Asp Ser Tyr 130 135 140 Lys Lys Ser His Asp Pro Ala Phe
Val Thr Leu Arg Asp Glu Leu Ser 145 150 155 160 Lys Glu Ile Asp Ala
Val Glu Glu Lys Ala Arg Gly Leu Arg Asp Arg 165 170 175 Ala Cys Asn
Asp Leu Tyr Glu Val Leu Arg Ser Asp Ser Glu Lys Phe 180 185 190 Arg
Glu Trp Leu Tyr Lys Glu Val Arg Ser Pro Glu Glu Ile Asn Asp 195 200
205 His Ala Ile Lys Asp Gly Met Cys Gly Tyr Glu Leu Val Lys Arg Glu
210 215 220 Leu Lys His Ala Asp Leu Leu Ile Cys Asn Tyr His His Val
Leu Asn 225 230 235 240 Pro Asp Ile Phe Ser Thr Val Leu Gly Trp Ile
Glu Lys Glu Pro Gln 245 250 255 Glu Thr Ile Val Ile Phe Asp Glu Ala
His Asn Leu Glu Ser Ala Ala 260 265 270 Arg Ser His Ser Ser Leu Ser
Leu Thr Glu His Ser Ile Glu Lys Ala 275 280 285 Ile Thr Glu Leu Glu
Ala Asn Leu Asp Leu Leu Ala Asp Asp Asn Ile 290 295 300 His Asn Leu
Phe Asn Ile Phe Leu Glu Val Ile Ser Asp Thr Tyr Asn 305 310 315 320
Ser Arg Phe Lys Phe Gly Glu Arg Glu Arg Val Arg Lys Asn Trp Tyr 325
330 335 Asp Ile Arg Ile Ser Asp Pro Tyr Glu Arg Asn Asp Ile Val Arg
Gly 340 345 350 Lys Phe Leu Arg Gln Ala Lys Gly Asp Phe Gly Glu Lys
Asp Asp Ile 355 360 365 Gln Ile Leu Leu Ser Glu Ala Ser Glu Leu Gly
Ala Lys Leu Asp Glu 370 375 380 Thr Tyr Arg Asp Gln Tyr Lys Lys Gly
Leu Ser Ser Val Met Lys Arg 385 390 395 400 Ser His Ile Arg Tyr Val
Ala Asp Phe Met Ser Ala Tyr Ile Glu Leu 405 410 415 Ser His Asn Leu
Asn Tyr Tyr Pro Ile Leu Asn Val Arg Arg Asp Met 420 425 430 Asn Asp
Glu Ile Tyr Gly Arg Val Glu Leu Phe Thr Cys Ile Pro Lys 435 440 445
Asn Val Thr Glu Pro Leu Phe Asn Ser Leu Phe Ser Val Ile Leu Met 450
455 460 Ser Ala Thr Leu His Pro Phe Glu Met Val Lys Lys Thr Leu Gly
Ile 465 470 475 480 Thr Arg Asp Thr Cys Glu Met Ser Tyr Gly Thr Ser
Phe Pro Glu Glu 485 490 495 Lys Arg Leu Ser Ile Ala Val Ser Ile Pro
Pro Leu Phe Ala Lys Asn 500 505 510 Arg Asp Asp Arg His Val Thr Glu
Leu Leu Glu Gln Val Leu Leu Asp 515 520 525 Ser Ile Glu Asn Ser Lys
Gly Asn Val Ile Leu Phe Phe Gln Ser Ala 530 535 540 Phe Glu Ala Lys
Arg Tyr Tyr Ser Lys Ile Glu Pro Leu Val Asn Val 545 550 555 560 Pro
Val Phe Leu Asp Glu Val Gly Ile Ser Ser Gln Asp Val Arg Glu 565 570
575 Glu Phe Phe Ser Ile Gly Glu Glu Asn Gly Lys Ala Val Leu Leu Ser
580 585 590 Tyr Leu Trp Gly Thr Leu Ser Glu Gly Ile Asp Tyr Arg Asp
Gly Arg 595 600 605 Gly Arg Thr Val Ile Ile Ile Gly Val Gly Tyr Pro
Ala Leu Asn Asp 610 615 620 Arg Met Asn Ala Val Glu Ser Ala Tyr Asp
His Val Phe Gly Tyr Gly 625 630 635 640 Ala Gly Trp Glu Phe Ala Ile
Gln Val Pro Thr Ile Arg Lys Ile Arg 645 650 655 Gln Ala Met Gly Arg
Val Val Arg Ser Pro Thr Asp Tyr Gly Ala Arg 660 665 670 Ile Leu Leu
Asp Gly Arg Phe Leu Thr Asp Ser Lys Lys Arg Phe Gly 675 680 685 Lys
Phe Ser Val Phe Glu Val Phe Pro Pro Ala Glu Arg Ser Glu Phe 690 695
700 Val Asp Val Asp Pro Glu Lys Val Lys Tyr Ser Leu Met Asn Phe Phe
705 710 715 720 Met Asp Asn Asp Glu Gln 725 24439PRTEnterobacteria
phage T4 24Met Thr Phe Asp Asp Leu Thr Glu Gly Gln Lys Asn Ala Phe
Asn Ile 1 5 10 15 Val Met Lys Ala Ile Lys Glu Lys Lys His His Val
Thr Ile Asn Gly 20 25 30 Pro Ala Gly Thr Gly Lys Thr Thr Leu Thr
Lys Phe Ile Ile Glu Ala 35 40 45 Leu Ile Ser Thr Gly Glu Thr Gly
Ile Ile Leu Ala Ala Pro Thr His 50 55 60 Ala Ala Lys Lys Ile Leu
Ser Lys Leu Ser Gly Lys Glu Ala Ser Thr 65 70 75 80 Ile His Ser Ile
Leu Lys Ile Asn Pro Val Thr Tyr Glu Glu Asn Val 85 90 95 Leu Phe
Glu Gln Lys Glu Val Pro Asp Leu Ala Lys Cys Arg Val Leu 100 105 110
Ile Cys Asp Glu Val Ser Met Tyr Asp Arg Lys Leu Phe Lys Ile Leu 115
120 125 Leu Ser Thr Ile Pro Pro Trp Cys Thr Ile Ile Gly Ile Gly Asp
Asn 130 135 140 Lys Gln Ile Arg Pro Val Asp Pro Gly Glu Asn Thr Ala
Tyr Ile Ser 145 150 155 160 Pro Phe Phe Thr His Lys Asp Phe Tyr Gln
Cys Glu Leu Thr Glu Val 165 170 175 Lys Arg Ser Asn Ala Pro Ile Ile
Asp Val Ala Thr Asp Val Arg Asn 180 185 190 Gly Lys Trp Ile Tyr Asp
Lys Val Val Asp Gly His Gly Val Arg Gly 195 200 205 Phe Thr Gly Asp
Thr Ala Leu Arg Asp Phe Met Val Asn Tyr Phe Ser 210 215 220 Ile Val
Lys Ser Leu Asp Asp Leu Phe Glu Asn Arg Val Met Ala Phe 225 230 235
240 Thr Asn Lys Ser Val Asp Lys Leu Asn Ser Ile Ile Arg Lys Lys Ile
245 250 255 Phe Glu Thr Asp Lys Asp Phe Ile Val Gly Glu Ile Ile Val
Met Gln 260 265 270 Glu Pro Leu Phe Lys Thr Tyr Lys Ile Asp Gly Lys
Pro Val Ser Glu 275 280 285 Ile Ile Phe Asn Asn Gly Gln Leu Val Arg
Ile Ile Glu Ala Glu Tyr 290 295 300 Thr Ser Thr Phe Val Lys Ala Arg
Gly Val Pro Gly Glu Tyr Leu Ile 305 310 315 320 Arg His Trp Asp Leu
Thr Val Glu Thr Tyr Gly Asp Asp Glu Tyr Tyr 325 330 335 Arg Glu Lys
Ile Lys Ile Ile Ser Ser Asp Glu Glu Leu Tyr Lys Phe 340 345 350 Asn
Leu Phe Leu Gly Lys Thr Ala Glu Thr Tyr Lys Asn Trp Asn Lys 355 360
365 Gly Gly Lys Ala Pro Trp Ser Asp Phe Trp Asp Ala Lys Ser Gln Phe
370 375 380 Ser Lys Val Lys Ala Leu Pro Ala Ser Thr Phe His Lys Ala
Gln Gly 385 390 395 400 Met Ser Val Asp Arg Ala Phe Ile Tyr Thr Pro
Cys Ile His Tyr Ala 405 410 415 Asp Val Glu Leu Ala Gln Gln Leu Leu
Tyr Val Gly Val Thr Arg Gly 420 425 430 Arg Tyr Asp Val Phe Tyr Val
435 25970PRTClostridium botulinum 25Met Leu Ser Val Ala Asn Val Arg
Ser Pro Ser Ala Ala Ala Ser Tyr 1 5 10 15 Phe Ala Ser Asp Asn Tyr
Tyr Ala Ser Ala Asp Ala Asp Arg Ser Gly 20 25 30 Gln Trp Ile Gly
Asp Gly Ala Lys Arg Leu Gly Leu Glu Gly Lys Val 35 40 45 Glu Ala
Arg Ala Phe Asp Ala Leu Leu Arg Gly Glu Leu Pro Asp Gly 50 55 60
Ser Ser Val Gly Asn Pro Gly Gln Ala His Arg Pro Gly Thr Asp Leu 65
70 75 80 Thr Phe Ser Val Pro Lys Ser Trp Ser Leu Leu Ala Leu Val
Gly Lys 85 90 95 Asp Glu Arg Ile Ile Ala Ala Tyr Arg Glu Ala Val
Val Glu Ala Leu 100 105 110 His Trp Ala Glu Lys Asn Ala Ala Glu Thr
Arg Val Val Glu Lys Gly 115 120 125 Met Val Val Thr Gln Ala Thr Gly
Asn Leu Ala Ile Gly Leu Phe Gln 130 135 140 His Asp Thr Asn Arg Asn
Gln Glu Pro Asn Leu His Phe His Ala Val 145 150 155 160 Ile Ala Asn
Val Thr Gln Gly Lys Asp Gly Lys Trp Arg Thr Leu Lys 165 170 175 Asn
Asp Arg Leu Trp Gln Leu Asn Thr Thr Leu Asn Ser Ile Ala Met 180 185
190 Ala Arg Phe Arg Val Ala Val Glu Lys Leu Gly Tyr Glu Pro Gly Pro
195 200 205 Val Leu Lys His Gly Asn Phe Glu Ala Arg Gly Ile Ser Arg
Glu Gln 210 215 220 Val Met Ala Phe Ser Thr Arg Arg Lys Glu Val Leu
Glu Ala Arg Arg 225 230 235 240 Gly Pro Gly Leu Asp Ala Gly Arg Ile
Ala Ala Leu Asp Thr Arg Ala 245 250 255 Ser Lys Glu Gly Ile Glu Asp
Arg Ala Thr Leu Ser Lys Gln Trp Ser 260 265 270 Glu Ala Ala Gln Ser
Ile Gly Leu Asp Leu Lys Pro Leu Val Asp Arg 275 280 285 Ala Arg Thr
Lys Ala Leu Gly Gln Gly Met Glu Ala Thr Arg Ile Gly 290 295 300 Ser
Leu Val Glu Arg Gly Arg Ala Trp Leu Ser Arg Phe Ala Ala His 305 310
315 320 Val Arg Gly Asp Pro Ala Asp Pro Leu Val Pro Pro Ser Val Leu
Lys 325 330 335 Gln Asp Arg Gln Thr Ile Ala Ala Ala Gln Ala Val Ala
Ser Ala Val 340 345 350 Arg His Leu Ser Gln Arg Glu Ala Ala Phe Glu
Arg Thr Ala Leu Tyr 355 360 365 Lys Ala Ala Leu Asp Phe Gly Leu Pro
Thr Thr Ile Ala Asp Val Glu 370 375 380 Lys Arg Thr Arg Ala Leu Val
Arg Ser Gly Asp Leu Ile Ala Gly Lys 385 390 395 400 Gly Glu His Lys
Gly Trp Leu Ala Ser Arg Asp Ala Val Val Thr Glu 405 410 415 Gln Arg
Ile Leu Ser Glu Val Ala Ala Gly Lys Gly Asp Ser Ser Pro 420 425 430
Ala Ile Thr Pro Gln Lys Ala Ala Ala Ser Val Gln Ala Ala Ala Leu 435
440 445 Thr Gly Gln Gly Phe Arg Leu Asn Glu Gly Gln Leu Ala Ala Ala
Arg 450 455 460 Leu Ile Leu Ile Ser Lys Asp Arg Thr Ile Ala Val Gln
Gly Ile Ala 465 470 475 480 Gly Ala Gly Lys Ser Ser Val Leu Lys Pro
Val Ala Glu Val Leu Arg 485 490 495 Asp Glu Gly His Pro Val Ile Gly
Leu Ala Ile Gln Asn Thr Leu Val 500 505 510 Gln Met Leu Glu Arg Asp
Thr Gly Ile Gly Ser Gln Thr Leu Ala Arg 515 520 525 Phe Leu Gly Gly
Trp Asn Lys Leu Leu Asp Asp Pro Gly Asn Val Ala 530 535 540 Leu Arg
Ala Glu Ala Gln Ala Ser Leu Lys Asp His Val Leu Val Leu 545 550 555
560 Asp Glu Ala Ser Met Val Ser Asn Glu Asp Lys Glu Lys Leu Val Arg
565 570 575 Leu Ala Asn Leu Ala Gly Val His Arg Leu Val Leu Ile Gly
Asp Arg 580 585 590 Lys Gln Leu Gly Ala Val Asp Ala Gly Lys Pro Phe
Ala Leu Leu Gln 595 600 605 Arg Ala Gly Ile Ala Arg Ala Glu Met Ala
Thr Asn Leu Arg Ala Arg 610 615 620 Asp Pro Val Val Arg Glu Ala Gln
Ala Ala Ala Gln Ala Gly Asp Val 625 630 635 640 Arg Lys Ala Leu Arg
His Leu Lys Ser His Thr Val Glu Ala Arg Gly 645 650 655 Asp Gly Ala
Gln Val Ala Ala Glu Thr Trp Leu Ala Leu Asp Lys Glu 660 665 670 Thr
Arg Ala Arg Thr Ser Ile Tyr Ala Ser Gly Arg Ala Ile Arg Ser 675 680
685 Ala Val Asn Ala Ala Val Gln Gln Gly Leu Leu Ala Ser Arg Glu Ile
690 695 700 Gly Pro Ala Lys Met Lys Leu Glu Val Leu Asp Arg Val Asn
Thr Thr 705 710 715 720 Arg Glu Glu Leu Arg His Leu Pro Ala Tyr Arg
Ala Gly Arg Val Leu 725 730 735 Glu Val Ser Arg Lys Gln Gln Ala Leu
Gly Leu Phe Ile Gly Glu Tyr 740 745 750 Arg Val Ile Gly Gln Asp Arg
Lys Gly Lys Leu Val Glu Val Glu Asp 755 760 765 Lys Arg Gly Lys Arg
Phe Arg Phe Asp Pro Ala Arg Ile Arg Ala Gly 770 775 780 Lys Gly Asp
Asp Asn Leu Thr Leu Leu Glu Pro Arg Lys Leu Glu Ile 785 790 795 800
His Glu Gly Asp Arg Ile Arg Trp Thr Arg Asn Asp His Arg Arg Gly 805
810 815 Leu Phe Asn Ala Asp Gln Ala Arg Val Val Glu Ile Ala Asn Gly
Lys 820 825 830 Val Thr Phe Glu Thr Ser Lys Gly Asp Leu Val Glu Leu
Lys Lys Asp 835 840 845 Asp Pro Met Leu Lys Arg Ile Asp Leu Ala Tyr
Ala Leu Asn Val His 850 855 860 Met Ala Gln Gly Leu Thr Ser Asp Arg
Gly Ile Ala Val Met Asp Ser 865 870 875 880 Arg Glu Arg Asn Leu Ser
Asn Gln Lys Thr Phe Leu Val Thr Val Thr 885 890 895 Arg Leu Arg Asp
His Leu Thr Leu Val Val Asp Ser Ala Asp Lys Leu 900 905 910 Gly Ala
Ala Val Ala Arg Asn Lys Gly Glu Lys Ala Ser Ala Ile Glu 915 920 925
Val Thr Gly Ser Val Lys Pro Thr Ala Thr Lys Gly Ser Gly Val Asp 930
935 940 Gln Pro Lys Ser Val Glu Ala Asn Lys Ala Glu Lys Glu Leu Thr
Arg 945 950 955 960 Ser Lys Ser Lys Thr Leu Asp Phe Gly Ile 965 970
2643DNAArtificial SequenceSynthetic Polynucleotide 26tttttttttt
cttttttttc ttttttggtt ggttgttggt tgg 432720DNAArtificial
SequenceSynthetic Polynucleotide 27tttttttttt cttttttttt
202817DNAArtificial SequenceSynthetic Polynucleotide 28ggttggttgt
tggttgg 172923DNAArtificial SequenceSynthetic Polynucleotide
29ttaatgctaa tcgtgatagg ggt 233028DNAArtificial SequenceSynthetic
Polynucleotide 30gttctactaa accgtgtcaa tcagtgtc
28313560DNAArtificial SequenceSynthetic Polynucleotide 31gccatcagat
tgtgtttgtt agtcgctttt tttttttgga attttttttt tggaattttt 60tttttgcgct
aacaacctcc tgccgttttg cccgtgcata tcggtcacga acaaatctga
120ttactaaaca cagtagcctg gatttgttct atcagtaatc gaccttattc
ctaattaaat 180agagcaaatc cccttattgg gggtaagaca tgaagatgcc
agaaaaacat gacctgttgg 240ccgccattct cgcggcaaag gaacaaggca
tcggggcaat ccttgcgttt gcaatggcgt 300accttcgcgg cagatataat
ggcggtgcgt ttacaaaaac agtaatcgac gcaacgatgt 360gcgccattat
cgcctagttc attcgtgacc ttctcgactt cgccggacta agtagcaatc
420tcgcttatat aacgagcgtg tttatcggct acatcggtac tgactcgatt
ggttcgctta 480tcaaacgctt cgctgctaaa aaagccggag tagaagatgg
tagaaatcaa taatcaacgt 540aaggcgttcc tcgatatgct ggcgtggtcg
gagggaactg ataacggacg tcagaaaacc 600agaaatcatg gttatgacgt
cattgtaggc ggagagctat ttactgatta ctccgatcac 660cctcgcaaac
ttgtcacgct aaacccaaaa ctcaaatcaa caggcgccgg acgctaccag
720cttctttccc gttggtggga tgcctaccgc aagcagcttg gcctgaaaga
cttctctccg 780aaaagtcagg acgctgtggc attgcagcag attaaggagc
gtggcgcttt acctatgatt 840gatcgtggtg atatccgtca ggcaatcgac
cgttgcagca atatctgggc ttcactgccg 900ggcgctggtt atggtcagtt
cgagcataag gctgacagcc tgattgcaaa attcaaagaa 960gcgggcggaa
cggtcagaga gattgatgta tgagcagagt caccgcgatt atctccgctc
1020tggttatctg catcatcgtc tgcctgtcat gggctgttaa tcattaccgt
gataacgcca 1080ttacctacaa agcccagcgc gacaaaaatg ccagagaact
gaagctggcg aacgcggcaa 1140ttactgacat gcagatgcgt cagcgtgatg
ttgctgcgct cgatgcaaaa tacacgaagg 1200agttagctga tgctaaagct
gaaaatgatg ctctgcgtga tgatgttgcc gctggtcgtc 1260gtcggttgca
catcaaagca gtctgtcagt cagtgcgtga agccaccacc gcctccggcg
1320tggataatgc agcctccccc cgactggcag acaccgctga acgggattat
ttcaccctca 1380gagagaggct gatcactatg caaaaacaac tggaaggaac
ccagaagtat attaatgagc 1440agtgcagata gagttgccca tatcgatggg
caactcatgc aattattgtg agcaatacac 1500acgcgcttcc agcggagtat
aaatgcctaa agtaataaaa ccgagcaatc catttacgaa 1560tgtttgctgg
gtttctgttt taacaacatt ttctgcgccg ccacaaattt tggctgcatc
1620gacagttttc ttctgcccaa ttccagaaac gaagaaatga tgggtgatgg
tttcctttgg 1680tgctactgct gccggtttgt tttgaacagt aaacgtctgt
tgagcacatc ctgtaataag 1740cagggccagc gcagtagcga gtagcatttt
tttcatggtg ttattcccga tgctttttga 1800agttcgcaga atcgtatgtg
tagaaaatta aacaaaccct aaacaatgag ttgaaatttc 1860atattgttaa
tatttattaa tgtatgtcag gtgcgatgaa tcgtcattgt attcccggat
1920taactatgtc cacagccctg acggggaact tctctgcggg agtgtccggg
aataattaaa 1980acgatgcaca cagggtttag cgcgtacacg tattgcatta
tgccaacgcc ccggtgctga 2040cacggaagaa accggacgtt atgatttagc
gtggaaagat ttgtgtagtg ttctgaatgc 2100tctcagtaaa tagtaatgaa
ttatcaaagg tatagtaata tcttttatgt tcatggatat 2160ttgtaaccca
tcggaaaact cctgctttag caagattttc cctgtattgc tgaaatgtga
2220tttctcttga tttcaaccta tcataggacg tttctataag atgcgtgttt
cttgagaatt 2280taacatttac aaccttttta agtcctttta ttaacacggt
gttatcgttt tctaacacga 2340tgtgaatatt atctgtggct agatagtaaa
tataatgtga gacgttgtga cgttttagtt 2400cagaataaaa caattcacag
tctaaatctt ttcgcacttg atcgaatatt tctttaaaaa 2460tggcaacctg
agccattggt aaaaccttcc atgtgatacg agggcgcgta gtttgcatta
2520tcgtttttat cgtttcaatc tggtctgacc tccttgtgtt ttgttgatga
tttatgtcaa 2580atattaggaa tgttttcact taatagtatt ggttgcgtaa
caaagtgcgg tcctgctggc 2640attctggagg gaaatacaac cgacagatgt
atgtaaggcc aacgtgctca aatcttcata 2700cagaaagatt tgaagtaata
ttttaaccgc tagatgaaga gcaagcgcat ggagcgacaa 2760aatgaataaa
gaacaatctg ctgatgatcc ctccgtggat ctgattcgtg taaaaaatat
2820gcttaatagc accatttcta tgagttaccc tgatgttgta attgcatgta
tagaacataa 2880ggtgtctctg gaagcattca gagcaattga ggcagcgttg
gtgaagcacg ataataatat 2940gaaggattat tccctggtgg ttgactgatc
accataactg ctaatcattc aaactattta 3000gtctgtgaca gagccaacac
gcagtctgtc actgtcagga aagtggtaaa actgcaactc 3060aattactgca
atgccctcgt aattaagtga atttacaata tcgtcctgtt cggagggaag
3120aacgcgggat gttcattctt catcactttt aattgatgta tatgctctct
tttctgacgt 3180tagtctccga cggcaggctt caatgaccca ggctgagaaa
ttcccggacc ctttttgctc 3240aagagcgatg ttaatttgtt caatcatttg
gttaggaaag cggatgttgc gggttgttgt 3300tctgcgggtt ctgttcttcg
ttgacatgag gttgccccgt attcagtgtc gctgatttgt 3360attgtctgaa
gttgttttta cgttaagttg atgcagatca attaatacga tacctgcgtc
3420ataattgatt atttgacgtg gtttgatggc ctccacgcac gttgtgatat
gtagatgata 3480atcattatca ctttacgggt cctttccggt gaaaaaaaag
gtaccaaaaa aaacatcgtc 3540gtgagtagtg aaccgtaagc
35603285DNAArtificial SequenceSynthetic Polynucleotide 32gccatcagat
tgtgtttgtt agtcgctttt tttttttgga attttttttt tggaattttt 60tttttgcgct
aacaacctcc tgccg 853372DNAArtificial SequenceSynthetic
Polynucleotide 33gcttacggtt cactactcac gacgatgttt tttttggtac
cttttttttc accggaaagg 60acccgtaaag tg 723446DNAArtificial
SequenceSynthetic Polynucleotide 34tttttttttt tttttttttt tttttttttt
tttttttttt tttttt 463527DNAArtificial SequenceSynthetic
Polynucleotide 35ggttgtttct gttggtgctg atattgc 273627DNAArtificial
SequenceSynthetic Polynucleotide 36gccatcagat tgtgtttgtt agtcgct
273727DNAArtificial SequenceSynthetic Polynucleotide 37acactgattg
acacggttta gtagaac 273827DNAArtificial SequenceSynthetic
Polynucleotide 38gcttacggtt cactactcac gacgatg
27393587DNAArtificial SequenceSynthetic Polynucleotide 39gccatcagat
tgtgtttgtt agtcgctgcc atcagattgt gtttgttagt cgcttttttt 60ttttggaatt
ttttttttgg aatttttttt ttgcgctaac aacctcctgc cgttttgccc
120gtgcatatcg gtcacgaaca aatctgatta ctaaacacag tagcctggat
ttgttctatc 180agtaatcgac cttattccta attaaataga gcaaatcccc
ttattggggg taagacatga 240agatgccaga aaaacatgac ctgttggccg
ccattctcgc ggcaaaggaa caaggcatcg 300gggcaatcct tgcgtttgca
atggcgtacc ttcgcggcag atataatggc ggtgcgttta 360caaaaacagt
aatcgacgca acgatgtgcg ccattatcgc ctagttcatt cgtgaccttc
420tcgacttcgc cggactaagt agcaatctcg cttatataac gagcgtgttt
atcggctaca 480tcggtactga ctcgattggt tcgcttatca aacgcttcgc
tgctaaaaaa gccggagtag 540aagatggtag aaatcaataa tcaacgtaag
gcgttcctcg atatgctggc gtggtcggag 600ggaactgata acggacgtca
gaaaaccaga aatcatggtt atgacgtcat tgtaggcgga 660gagctattta
ctgattactc cgatcaccct cgcaaacttg tcacgctaaa cccaaaactc
720aaatcaacag gcgccggacg ctaccagctt ctttcccgtt ggtgggatgc
ctaccgcaag 780cagcttggcc tgaaagactt ctctccgaaa agtcaggacg
ctgtggcatt gcagcagatt 840aaggagcgtg gcgctttacc tatgattgat
cgtggtgata tccgtcaggc aatcgaccgt 900tgcagcaata tctgggcttc
actgccgggc gctggttatg gtcagttcga gcataaggct 960gacagcctga
ttgcaaaatt caaagaagcg ggcggaacgg tcagagagat tgatgtatga
1020gcagagtcac cgcgattatc tccgctctgg ttatctgcat catcgtctgc
ctgtcatggg 1080ctgttaatca ttaccgtgat aacgccatta cctacaaagc
ccagcgcgac aaaaatgcca 1140gagaactgaa gctggcgaac gcggcaatta
ctgacatgca gatgcgtcag cgtgatgttg 1200ctgcgctcga tgcaaaatac
acgaaggagt tagctgatgc taaagctgaa aatgatgctc 1260tgcgtgatga
tgttgccgct ggtcgtcgtc ggttgcacat caaagcagtc tgtcagtcag
1320tgcgtgaagc caccaccgcc tccggcgtgg ataatgcagc ctccccccga
ctggcagaca 1380ccgctgaacg ggattatttc accctcagag agaggctgat
cactatgcaa aaacaactgg 1440aaggaaccca gaagtatatt aatgagcagt
gcagatagag ttgcccatat cgatgggcaa 1500ctcatgcaat tattgtgagc
aatacacacg cgcttccagc ggagtataaa tgcctaaagt 1560aataaaaccg
agcaatccat ttacgaatgt ttgctgggtt tctgttttaa caacattttc
1620tgcgccgcca caaattttgg ctgcatcgac agttttcttc tgcccaattc
cagaaacgaa 1680gaaatgatgg gtgatggttt cctttggtgc tactgctgcc
ggtttgtttt gaacagtaaa 1740cgtctgttga gcacatcctg taataagcag
ggccagcgca gtagcgagta gcattttttt 1800catggtgtta ttcccgatgc
tttttgaagt tcgcagaatc gtatgtgtag aaaattaaac 1860aaaccctaaa
caatgagttg aaatttcata ttgttaatat ttattaatgt atgtcaggtg
1920cgatgaatcg tcattgtatt cccggattaa ctatgtccac agccctgacg
gggaacttct 1980ctgcgggagt gtccgggaat aattaaaacg atgcacacag
ggtttagcgc gtacacgtat 2040tgcattatgc caacgccccg gtgctgacac
ggaagaaacc ggacgttatg atttagcgtg 2100gaaagatttg tgtagtgttc
tgaatgctct cagtaaatag taatgaatta tcaaaggtat 2160agtaatatct
tttatgttca tggatatttg taacccatcg gaaaactcct gctttagcaa
2220gattttccct gtattgctga aatgtgattt ctcttgattt caacctatca
taggacgttt 2280ctataagatg cgtgtttctt gagaatttaa catttacaac
ctttttaagt ccttttatta 2340acacggtgtt atcgttttct aacacgatgt
gaatattatc tgtggctaga tagtaaatat 2400aatgtgagac gttgtgacgt
tttagttcag aataaaacaa ttcacagtct aaatcttttc 2460gcacttgatc
gaatatttct ttaaaaatgg caacctgagc cattggtaaa accttccatg
2520tgatacgagg gcgcgtagtt tgcattatcg tttttatcgt ttcaatctgg
tctgacctcc 2580ttgtgttttg ttgatgattt atgtcaaata ttaggaatgt
tttcacttaa tagtattggt 2640tgcgtaacaa agtgcggtcc tgctggcatt
ctggagggaa atacaaccga cagatgtatg 2700taaggccaac gtgctcaaat
cttcatacag aaagatttga agtaatattt taaccgctag 2760atgaagagca
agcgcatgga gcgacaaaat gaataaagaa caatctgctg atgatccctc
2820cgtggatctg attcgtgtaa aaaatatgct taatagcacc atttctatga
gttaccctga 2880tgttgtaatt gcatgtatag aacataaggt gtctctggaa
gcattcagag caattgaggc 2940agcgttggtg aagcacgata ataatatgaa
ggattattcc ctggtggttg actgatcacc 3000ataactgcta atcattcaaa
ctatttagtc tgtgacagag ccaacacgca gtctgtcact 3060gtcaggaaag
tggtaaaact gcaactcaat tactgcaatg ccctcgtaat taagtgaatt
3120tacaatatcg tcctgttcgg agggaagaac gcgggatgtt cattcttcat
cacttttaat 3180tgatgtatat gctctctttt ctgacgttag tctccgacgg
caggcttcaa tgacccaggc 3240tgagaaattc ccggaccctt tttgctcaag
agcgatgtta atttgttcaa tcatttggtt 3300aggaaagcgg atgttgcggg
ttgttgttct gcgggttctg ttcttcgttg acatgaggtt 3360gccccgtatt
cagtgtcgct gatttgtatt gtctgaagtt gtttttacgt taagttgatg
3420cagatcaatt aatacgatac ctgcgtcata attgattatt tgacgtggtt
tgatggcctc 3480cacgcacgtt gtgatatgta gatgataatc attatcactt
tacgggtcct ttccggtgaa 3540aaaaaaggta ccaaaaaaaa catcgtcgtg
agtagtgaac cgtaagc 3587403560DNAArtificial SequenceSynthetic
Polynucleotide 40gcttacggtt cactactcac gacgatgttt tttttggtac
cttttttttc accggaaagg 60acccgtaaag tgataatgat tatcatctac atatcacaac
gtgcgtggag gccatcaaac 120cacgtcaaat aatcaattat gacgcaggta
tcgtattaat tgatctgcat caacttaacg 180taaaaacaac ttcagacaat
acaaatcagc gacactgaat acggggcaac ctcatgtcaa 240cgaagaacag
aacccgcaga acaacaaccc gcaacatccg ctttcctaac caaatgattg
300aacaaattaa catcgctctt gagcaaaaag ggtccgggaa tttctcagcc
tgggtcattg 360aagcctgccg tcggagacta acgtcagaaa agagagcata
tacatcaatt aaaagtgatg 420aagaatgaac atcccgcgtt cttccctccg
aacaggacga tattgtaaat tcacttaatt 480acgagggcat tgcagtaatt
gagttgcagt tttaccactt tcctgacagt gacagactgc 540gtgttggctc
tgtcacagac taaatagttt gaatgattag cagttatggt gatcagtcaa
600ccaccaggga ataatccttc atattattat cgtgcttcac caacgctgcc
tcaattgctc 660tgaatgcttc cagagacacc ttatgttcta tacatgcaat
tacaacatca gggtaactca 720tagaaatggt gctattaagc atatttttta
cacgaatcag atccacggag ggatcatcag 780cagattgttc tttattcatt
ttgtcgctcc atgcgcttgc tcttcatcta gcggttaaaa 840tattacttca
aatctttctg tatgaagatt tgagcacgtt ggccttacat acatctgtcg
900gttgtatttc cctccagaat gccagcagga ccgcactttg ttacgcaacc
aatactatta 960agtgaaaaca ttcctaatat ttgacataaa tcatcaacaa
aacacaagga ggtcagacca 1020gattgaaacg ataaaaacga taatgcaaac
tacgcgccct cgtatcacat ggaaggtttt 1080accaatggct caggttgcca
tttttaaaga aatattcgat caagtgcgaa aagatttaga 1140ctgtgaattg
ttttattctg aactaaaacg tcacaacgtc tcacattata tttactatct
1200agccacagat aatattcaca tcgtgttaga aaacgataac accgtgttaa
taaaaggact 1260taaaaaggtt gtaaatgtta aattctcaag aaacacgcat
cttatagaaa cgtcctatga 1320taggttgaaa tcaagagaaa tcacatttca
gcaatacagg gaaaatcttg ctaaagcagg 1380agttttccga tgggttacaa
atatccatga acataaaaga tattactata cctttgataa 1440ttcattacta
tttactgaga gcattcagaa cactacacaa atctttccac gctaaatcat
1500aacgtccggt ttcttccgtg tcagcaccgg ggcgttggca taatgcaata
cgtgtacgcg 1560ctaaaccctg tgtgcatcgt tttaattatt cccggacact
cccgcagaga agttccccgt 1620cagggctgtg gacatagtta atccgggaat
acaatgacga ttcatcgcac ctgacataca 1680ttaataaata ttaacaatat
gaaatttcaa ctcattgttt agggtttgtt taattttcta 1740cacatacgat
tctgcgaact tcaaaaagca tcgggaataa caccatgaaa aaaatgctac
1800tcgctactgc gctggccctg cttattacag gatgtgctca acagacgttt
actgttcaaa 1860acaaaccggc agcagtagca ccaaaggaaa ccatcaccca
tcatttcttc gtttctggaa 1920ttgggcagaa gaaaactgtc gatgcagcca
aaatttgtgg cggcgcagaa aatgttgtta 1980aaacagaaac ccagcaaaca
ttcgtaaatg gattgctcgg ttttattact ttaggcattt 2040atactccgct
ggaagcgcgt gtgtattgct cacaataatt gcatgagttg cccatcgata
2100tgggcaactc tatctgcact gctcattaat atacttctgg gttccttcca
gttgtttttg 2160catagtgatc agcctctctc tgagggtgaa ataatcccgt
tcagcggtgt ctgccagtcg 2220gggggaggct gcattatcca cgccggaggc
ggtggtggct tcacgcactg actgacagac 2280tgctttgatg tgcaaccgac
gacgaccagc ggcaacatca tcacgcagag catcattttc 2340agctttagca
tcagctaact ccttcgtgta ttttgcatcg agcgcagcaa catcacgctg
2400acgcatctgc atgtcagtaa ttgccgcgtt cgccagcttc agttctctgg
catttttgtc 2460gcgctgggct ttgtaggtaa tggcgttatc acggtaatga
ttaacagccc atgacaggca 2520gacgatgatg cagataacca gagcggagat
aatcgcggtg actctgctca tacatcaatc 2580tctctgaccg ttccgcccgc
ttctttgaat tttgcaatca ggctgtcagc cttatgctcg 2640aactgaccat
aaccagcgcc cggcagtgaa gcccagatat tgctgcaacg gtcgattgcc
2700tgacggatat caccacgatc aatcataggt aaagcgccac gctccttaat
ctgctgcaat 2760gccacagcgt cctgactttt cggagagaag tctttcaggc
caagctgctt gcggtaggca 2820tcccaccaac gggaaagaag ctggtagcgt
ccggcgcctg ttgatttgag ttttgggttt 2880agcgtgacaa gtttgcgagg
gtgatcggag taatcagtaa atagctctcc gcctacaatg 2940acgtcataac
catgatttct ggttttctga cgtccgttat cagttccctc cgaccacgcc
3000agcatatcga ggaacgcctt acgttgatta ttgatttcta ccatcttcta
ctccggcttt 3060tttagcagcg aagcgtttga taagcgaacc aatcgagtca
gtaccgatgt agccgataaa 3120cacgctcgtt atataagcga gattgctact
tagtccggcg aagtcgagaa ggtcacgaat 3180gaactaggcg ataatggcgc
acatcgttgc gtcgattact gtttttgtaa acgcaccgcc 3240attatatctg
ccgcgaaggt acgccattgc aaacgcaagg attgccccga tgccttgttc
3300ctttgccgcg agaatggcgg ccaacaggtc atgtttttct ggcatcttca
tgtcttaccc 3360ccaataaggg gatttgctct atttaattag gaataaggtc
gattactgat agaacaaatc 3420caggctactg tgtttagtaa tcagatttgt
tcgtgaccga tatgcacggg caaaacggca 3480ggaggttgtt agcgcaaaaa
aaaaattcca aaaaaaaaat tccaaaaaaa aaaagcgact 3540aacaaacaca
atctgatggc 35604129DNAArtificial SequenceSynthetic Polynucleotide
41gcaatatcag caccaacaga aacaacctt 2942103DNAArtificial
SequenceSynthetic Polynucleotide 42tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt 60tttttttttt ttttttggtt gtttctgttg
gtgctgatat tgc 10343606DNAArtificial SequenceSynthetic
Polynucleotide 43gccatcagat tgtgtttgtt agtcgctttt tttttttgga
attttttttt tggaattttt 60tttttgacgc tcagtaatgt gacgatagct gaaaactgta
cgataaacgg tacgctgagg 120gcggaaaaaa tcgtcgggga cattgtaaag
gcggcgagcg cggcttttcc gcgccagcgt 180gaaagcagtg tggactggcc
gtcaggtacc cgtactgtca ccgtgaccga tgaccatcct 240tttgatcgcc
agatagtggt gcttccgctg acgtttcgcg gaagtaagcg tactgtcagc
300ggcaggacaa cgtattcgat gtgttatctg aaagtactga tgaacggtgc
ggtgatttat 360gatggcgcgg cgaacgaggc ggtacaggtg ttctcccgta
ttgttgacat gccagcgggt 420cggggaaacg tgatcctgac gttcacgctt
acgtccacac ggcattcggc agatattccg 480ccgtatacgt ttgccagcga
tgtgcaggtt atggtgatta agaaacaggc gctgggcatc 540agcgtggtct
gagtgtgaaa aaaaaggtac caaaaaaaac atcgtcgtga gtagtgaacc 600gtaagc
60644606DNAArtificial SequenceSynthetic Polynucleotide 44gcttacggtt
cactactcac gacgatgttt tttttggtac cttttttttc acactcagac 60cacgctgatg
cccagcgcct gtttcttaat caccataacc tgcacatcgc tggcaaacgt
120atacggcgga atatctgccg aatgccgtgt ggacgtaagc gtgaacgtca
ggatcacgtt 180tccccgaccc gctggcatgt caacaatacg ggagaacacc
tgtaccgcct cgttcgccgc 240gccatcataa atcaccgcac cgttcatcag
tactttcaga taacacatcg aatacgttgt 300cctgccgctg acagtacgct
tacttccgcg aaacgtcagc ggaagcacca ctatctggcg 360atcaaaagga
tggtcatcgg tcacggtgac agtacgggta cctgacggcc agtccacact
420gctttcacgc tggcgcggaa aagccgcgct cgccgccttt acaatgtccc
cgacgatttt 480ttccgccctc agcgtaccgt ttatcgtaca gttttcagct
atcgtcacat tactgagcgt 540caaaaaaaaa attccaaaaa aaaaattcca
aaaaaaaaaa gcgactaaca aacacaatct 600gatggc 6064528DNAArtificial
SequenceSynthetic Polynucleotide 45gcaatatcag caccaacaga aacaacct
284610DNAArtificial SequenceSynthetic Polynucleotide 46tttttttttt
10473560DNAArtificial SequenceSynthetic Polynucleotide 47gcttacggtt
cactactcac gacgatgttt tttttggtac cttttttttc accggaaagg 60acccgtaaag
tgataatgat tatcatctac atatcacaac gtgcgtggag gccatcaaac
120cacgtcaaat aatcaattat gacgcaggta tcgtattaat tgatctgcat
caacttaacg 180taaaaacaac ttcagacaat acaaatcagc gacactgaat
acggggcaac ctcatgtcaa 240cgaagaacag aacccgcaga acaacaaccc
gcaacatccg ctttcctaac caaatgattg 300aacaaattaa catcgctctt
gagcaaaaag ggtccgggaa tttctcagcc tgggtcattg 360aagcctgccg
tcggagacta acgtcagaaa agagagcata tacatcaatt aaaagtgatg
420aagaatgaac atcccgcgtt cttccctccg aacaggacga tattgtaaat
tcacttaatt 480acgagggcat tgcagtaatt gagttgcagt tttaccactt
tcctgacagt gacagactgc 540gtgttggctc tgtcacagac taaatagttt
gaatgattag cagttatggt gatcagtcaa 600ccaccaggga ataatccttc
atattattat cgtgcttcac caacgctgcc tcaattgctc 660tgaatgcttc
cagagacacc ttatgttcta tacatgcaat tacaacatca gggtaactca
720tagaaatggt gctattaagc atatttttta cacgaatcag atccacggag
ggatcatcag 780cagattgttc tttattcatt ttgtcgctcc atgcgcttgc
tcttcatcta gcggttaaaa 840tattacttca aatctttctg tatgaagatt
tgagcacgtt ggccttacat acatctgtcg 900gttgtatttc cctccagaat
gccagcagga ccgcactttg ttacgcaacc aatactatta 960agtgaaaaca
ttcctaatat ttgacataaa tcatcaacaa aacacaagga ggtcagacca
1020gattgaaacg ataaaaacga taatgcaaac tacgcgccct cgtatcacat
ggaaggtttt 1080accaatggct caggttgcca tttttaaaga aatattcgat
caagtgcgaa aagatttaga 1140ctgtgaattg ttttattctg aactaaaacg
tcacaacgtc tcacattata tttactatct 1200agccacagat aatattcaca
tcgtgttaga aaacgataac accgtgttaa taaaaggact 1260taaaaaggtt
gtaaatgtta aattctcaag aaacacgcat cttatagaaa cgtcctatga
1320taggttgaaa tcaagagaaa tcacatttca gcaatacagg gaaaatcttg
ctaaagcagg 1380agttttccga tgggttacaa atatccatga acataaaaga
tattactata cctttgataa 1440ttcattacta tttactgaga gcattcagaa
cactacacaa atctttccac gctaaatcat 1500aacgtccggt ttcttccgtg
tcagcaccgg ggcgttggca taatgcaata cgtgtacgcg 1560ctaaaccctg
tgtgcatcgt tttaattatt cccggacact cccgcagaga agttccccgt
1620cagggctgtg gacatagtta atccgggaat acaatgacga ttcatcgcac
ctgacataca 1680ttaataaata ttaacaatat gaaatttcaa ctcattgttt
agggtttgtt taattttcta 1740cacatacgat tctgcgaact tcaaaaagca
tcgggaataa caccatgaaa aaaatgctac 1800tcgctactgc gctggccctg
cttattacag gatgtgctca acagacgttt actgttcaaa 1860acaaaccggc
agcagtagca ccaaaggaaa ccatcaccca tcatttcttc gtttctggaa
1920ttgggcagaa gaaaactgtc gatgcagcca aaatttgtgg cggcgcagaa
aatgttgtta 1980aaacagaaac ccagcaaaca ttcgtaaatg gattgctcgg
ttttattact ttaggcattt 2040atactccgct ggaagcgcgt gtgtattgct
cacaataatt gcatgagttg cccatcgata 2100tgggcaactc tatctgcact
gctcattaat atacttctgg gttccttcca gttgtttttg 2160catagtgatc
agcctctctc tgagggtgaa
ataatcccgt tcagcggtgt ctgccagtcg 2220gggggaggct gcattatcca
cgccggaggc ggtggtggct tcacgcactg actgacagac 2280tgctttgatg
tgcaaccgac gacgaccagc ggcaacatca tcacgcagag catcattttc
2340agctttagca tcagctaact ccttcgtgta ttttgcatcg agcgcagcaa
catcacgctg 2400acgcatctgc atgtcagtaa ttgccgcgtt cgccagcttc
agttctctgg catttttgtc 2460gcgctgggct ttgtaggtaa tggcgttatc
acggtaatga ttaacagccc atgacaggca 2520gacgatgatg cagataacca
gagcggagat aatcgcggtg actctgctca tacatcaatc 2580tctctgaccg
ttccgcccgc ttctttgaat tttgcaatca ggctgtcagc cttatgctcg
2640aactgaccat aaccagcgcc cggcagtgaa gcccagatat tgctgcaacg
gtcgattgcc 2700tgacggatat caccacgatc aatcataggt aaagcgccac
gctccttaat ctgctgcaat 2760gccacagcgt cctgactttt cggagagaag
tctttcaggc caagctgctt gcggtaggca 2820tcccaccaac gggaaagaag
ctggtagcgt ccggcgcctg ttgatttgag ttttgggttt 2880agcgtgacaa
gtttgcgagg gtgatcggag taatcagtaa atagctctcc gcctacaatg
2940acgtcataac catgatttct ggttttctga cgtccgttat cagttccctc
cgaccacgcc 3000agcatatcga ggaacgcctt acgttgatta ttgatttcta
ccatcttcta ctccggcttt 3060tttagcagcg aagcgtttga taagcgaacc
aatcgagtca gtaccgatgt agccgataaa 3120cacgctcgtt atataagcga
gattgctact tagtccggcg aagtcgagaa ggtcacgaat 3180gaactaggcg
ataatggcgc acatcgttgc gtcgattact gtttttgtaa acgcaccgcc
3240attatatctg ccgcgaaggt acgccattgc aaacgcaagg attgccccga
tgccttgttc 3300ctttgccgcg agaatggcgg ccaacaggtc atgtttttct
ggcatcttca tgtcttaccc 3360ccaataaggg gatttgctct atttaattag
gaataaggtc gattactgat agaacaaatc 3420caggctactg tgtttagtaa
tcagatttgt tcgtgaccga tatgcacggg caaaacggca 3480ggaggttgtt
agcgcaaaaa aaaaattcca aaaaaaaaat tccaaaaaaa aaaagcgact
3540aacaaacaca atctgatggc 3560
* * * * *
References